Inhibitors of β-amyloid protein production

ABSTRACT

This invention relates to compounds and pharmaceutical compositions, and methods for inhibiting or preventing the amyloid protein deposits in the brain which are associated with Alzheimer&#39;s disease and aged Down&#39;s syndrome patients. More particularly, it relates to the treatment of Alzheimer&#39;s disease.

This application is a national stage entry under 35 U.S.C. § 371 of anInternational Application No. PCT/US94/10679, filed Sep. 20, 1994, whichclaims the benefit of priority of European Patent Application No.93402398.7, filed Oct. 1, 1993.

TECHNICAL FIELD

This invention relates to compounds and pharmaceutical compositions, andmethods for inhibiting or preventing the amyloid protein deposits in thebrain. More particularly, the present invention relates to the treatmentof Alzheimer's disease.

BACKGROUND ART

It is estimated that over 5% of the U.S. population over 65 and over 15%of the U.S. population over 85 are affected by Alzheimer's disease.(Cross, A. J., Eur. J. Pharmacol. (1982) 82: 77 -80; Terry, R. D., etal., Ann. Neurol. (1983) 14: 497-506). It is believed that the principalcause of confinement of the elderly in long term care facilities is dueto this disease, and approximately 65% of those dying in skilled nursinghomes suffer from it.

Certain facts about the biochemical and metabolic phenomena associatedwith the presence of Alzheimer's disease are known. Two morphologicaland histopathological changes noted in Alzheimer's disease brains areneuro-fibrillary tangles (NFT) and amyloid deposits. Intraneuronalneurofibrillary tangles are present in other degenerative diseases aswell, but the presence of amyloid deposits both in the intraneuronalspaces (neuritic plaques) and in the surrounding microvasculature(vascular plaques) seems to be characteristic of Alzheimer's. Of these,the neuritic plaques seem to be the most prevalent (Price, D. L., etal., Drug Development Research (1985) 5: 59-68). Plaques are also seenin the brains of aged Down's Syndrome patients who develop Alzheimer'sdisease.

Plaque-rich brains of Alzheimer's patients have been used as a source toextract an approximately 4.2 kd "core" polypeptide, amyloid plaque coreprotein (APCP). This peptide was designated β-protein by (Glenner, G.,et al., Biochem. Biophys. Res. Commun. (1984) 120: 885-890). The aminoacid sequence of the amino-terminus was determined (Glenner, G., et al.,Biochem. Biophys. Res. Commun. (1984) 122: 1131-1135; Masters, C. L., etal., Proc. Natl. Acad. Sci USA (1985) 82: 4245-42259). The amino acidsequences reported by the two groups were identical, except that Glenneret al. reported a glutamine residue at position 11 for Alzheimer'sdisease cerebral vascular amyloid whereas Master et al. reportedglutamic acid at position 11. Also, the former authors reported that thecerebral vascular amyloid has a homogeneous amino-terminus, while thelatter authors reported heterogeneous amino-termini. Both groups showedthat the same peptide is found in the amyloid plaque cores and vascularamyloid of adult Down's syndrome-afflicted individuals and reportglutamic acid at position 11. Wong, C. W., et al. (Proc. Natl. Acad.Sci. USA (1985) 82: 8729-8732) showed that a synthetic peptide which washomologous to the first ten amino acids of the β-amyloid core proteindescribed by Masters (supra) was able to raise antibodies in mice andthat these antibodies could be used to stain not only amyloid-ladencerebral vessels, but also neuritic plaques. These results wereconfirmed by Allsop, D. et al., Neuroscience Letters (1986) 68: 252-256using antibodies directed against a synthetic peptide corresponding toamino acids 8-17. Thus, in general, the plaque protein found in variouslocations of the brain of Alzheimer's patients appears to be similar inimmuno-reactivity. It is highly insoluble, as shown by the inability toachieve solubilization in many commonly used denaturants, such asdetergents and chaotropic agents (Masters, supra, Allsop, D., et al.(supra)).

There are six known instances of disease-associated amyloid deposits inwhich the amyloid is produced from a precursor protein: for primaryamyloidosis, the precursor is an immunoglobulin light chain; forsecondary amyloidosis, the precursor is amyloid A protein; foramyloidosis, prealbumin or a variant thereof; for medullary carcinoma ofthyroid, a procalcitonin fragment; and for hereditary cerebralhemorrhage, gamma-trace fragment (See, e.g., Glenner, G. New EnglandJournal of Medicine (1980.) 302: 1283; Sletton, K., et al. Biochem J(1981) 195: 561; Benditt, et al. FEBS Lett(1971) 19:169; Sletton, K.,al., Eur J Biochem (1974) 41: 117; Sletton, K. J Exp Med (1976) 143:993). The foregoing is a partial list and there are at least a number ofadditional references with regard to procalcitonin fragment as aprecursor for the amyloid of the thyroid carcinoma.

It is believed, by analogy to other known instances ofdisease-associated amyloid deposits, that the β-amyloid core proteinassociated with Alzheimer's disease is formed from a precursor protein.A protein containing the β-amyloid core protein sequence within theframework of a larger protein was described by Kang, J., et al., (Nature(1987) 325: 733-736). The sequence of this protein was deduced from thesequence of a cDNA clone isolated from a human fetal brain tissue cDNAlibrary and consists of 695 amino acid residues wherein the aminoterminus of the β-amyloid core protein begins at position 597. A secondprecursor protein containing the β-amyloid sequence was predicted from acDNA clone isolated by Ponte, et al. (Nature (1988) 331: 525-527). ThecDNA clone isolated by Ponte et al. encoded a precursor protein which isidentical to that identified by Kang, et al., except that it contains anadditional 57-amino acid sequence inserted upstream of the β-amyloidcore protein sequence. The 57-amino acid insert sequence comprises afunctional domain which is highly homologous to a series of proteaseinhibitors known as the Kunitz-type serine protease inhibitors. Othershave characterized an additional amyloid precursor protein (SeeKitaguchi, et al., Nature (1988) 331: 530-532) which contains 770 aminoacids. The precursor identified by Kitaguchi is identical to that ofPonte et al., except that it contains an additional 19 amino acidsadjacent the 57-amino acid protease inhibitor domain. It is not knownthat these additional 19 amino acids provide any additionalfunctionality to the molecule. The various amyloid precursor proteinswhich have been identified from cDNA clones arise as the result ofalternative message splicing during transcription of a single amyloidprecursor gene.

It has been shown that the amyloid precursor proteins are processed bynormal cellular metabolism to produce the β-amyloid core protein (Haass,et al. Nature (1992) 359: 322-325; Shoji, et al. Science (1992) 258:126-129,; Seubert, et al. Nature (1992) 359: 325-327). It is unclear,however, if individuals with Alzheimer's disease produce higher amountsof the β-amyloid core protein, although it has been shown thatindividuals with Down's syndrome, who invariably develop Alzheimer'sdisease, express two-fold more B-amyloid precursor protein (Neve, et al.Neuron (1988) 1: 669-677). It is believed that the development ofamyloid plaques in Alzheimer's disease brains results from excessproduction and/or reduced clearance or sequestration of the β-amyloidprotein. Hence, if means could be devised for intervening in the processof plaque formation by preventing or inhibiting the processing of theprecursor to produce the amyloid plaque core protein, such means couldconstitute a method of treating or ameliorating the progression ofAlzheimer's disease. Until now, however, the processing of amyloidprecursor protein to produce the β-amyloid core protein has not beensufficiently understood to allow for effective therapeutic interventionin the process which results in amyloid deposition.

Numerous reports exist describing putative proteinases which arepurported to be responsible for generating the β-amyloid protein and/orAlzheimer's disease pathology. These putative proteinases include abroad spectrum of classes of enzymes, for example, serine, cysteine, andmetallo-proteinases. A number of β-amyloid forming proteinases have beenisolated and characterized. Some reported candidates includemulticatalytic proteinase (FEBS 304: 57-60 (1992) and FEBS Lett. 257:388-92 (1989)), mast cell chymase (J. Biol. Chem. 265: 3836-43 (1990),metallo-endopeptidase 24.15, Biochem. Biophys. Res. Commun. 185: 746-52(1992)), calcium-activated neutral proteinases (calpain) (J. Neurosci.)10: 2400-11 (1990), a calcium-activated serine proteinase (Biochem.Biophys. Res. Commun. 174: 790-96 (1991)), and prolyl-endopeptidase(FEBS Lett. 160: 131-34 (1990)). The physiological relevance for each ofthese candidate β-amyloid forming proteinases has not been demonstrated,for example, by concomitant inhibition of enzymatic activity withblocked β-amyloid protein formation.

Many proteinases have been reported as being altered in Alzheimer'sdisease brain tissue. For example, α-1-trypsin-like immunoreactivity hasbeen shown to be increased in Alzheimer's disease brain (Biochem.Biophys. Res. Comm. Vol. 193(2): 579-84 (1993)), three differentmetalloproteinases have been reported as elevated in Alzheimer's diseasebrain (J. Neurochem. Vol. 58: 983-92 (1992)), multicatalytic proteinasealterations have been observed (Neurosc. Res. Comm, Vol. 8(3): 185-90(1991)), abnormal cathepsin D and B immunoreactivity has been reported(Neurosc. Lett. 130: 195-98 (1991) and Proc. Natl. Acad. Sci. 87:3861-65 (1990)), and calcium-activated neutral proteinase (calpain) hasbeen variously shown to be decreased (Neurobio. of Aging, 11: 425-31(1990)), increased (Proc. Natl. Acad. Sci. USA 90: 2628-32 (1993)), orto be unaltered (J. Neurol. Sci, 102: 220-34 (1991)) in Alzheimer'sdisease brain tissue.

The foregoing demonstrates that, although there has been a tremendousamount of work reported in this area, there is no general consensus asto classes of proteinases which are effective in treating Alzheimer'sdisease or if these proteinases are altered upon contact withAlzheimer's disease brain tissue.

It is an object of the present invention to provide compounds andpharmaceutical compositions, and methods for inhibiting the productionof β-amyloid core protein and the formation of amyloid plaques in anindividual suffering from dementia of the Alzheimer's type.

It is a further object of the invention to provide pharmaceuticalcompositions, and methods for treating or ameliorating the progressionof Alzheimer's disease.

SUMMARY OF THE INVENTION

The present invention comprises compounds and the use of these compoundsfor treating conditions responsive to the inhibition or prevention ofproduction of β-amyloid core protein in a patient. Some examples ofthese conditions are senile dementia of the Alzheimer's type and agedDown's syndrome disease.

The compounds in Formula IA are used in the treatment of senile dementiaof the Alzheimer's type and aged Down's syndrome disease. However, someof these compounds have been previously disclosed for other uses.Therefore, Formula IB is also described which is a subset of Formula IAwhich is believed to describe compounds not previously disclosed.Formula IB differs from formula IA at the definition of X^(a) and X inthe provisos.

The compounds or the hydrate, stereoisomer, isostere or thepharmaceutically acceptable salt thereof are described in formulas IAwhich are used to treat senile dementia of the Alzheimer's type:

    K--P.sub.4 --P.sub.3 --P.sub.2 --NH--CH(R)--[C(═O)].sub.n --X (SEQ ID NO:1)                                                     IA

wherein

X is H, CHF₂, CF₃, CF₂ F₃, CF₂ CH₂ NHC(═O)R₁, CHFCH₂ NHC(═O)R₁, CF₂C(═O)W, C(═O)NHR₁, B(OH)₂, or C(═O)R₁, wherein W is NHCH₂ Si(C₁₋₆alkyl)₂ (Y), NHR₁ or R₁ ;

R is C₁₋₁₀ alkyl, benzyl, CH₂ Si(C₁₋₆ alkyl)₂ (Y), C₁₋₄ alkylene-O--R₁,CH₂ CH(CF₃)₂, CH₂ CH(CH₃)(CF₃), (CH₂)_(m) -naphthyl, or a substitutedbenzyl, the substitution being 1, 2 or 3 substituents independentlyselected from the group consisting of C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆alkoxyalkyl, benzyloxy, hydroxy, NHC(═NH)NH₂, NR₁ H, NO₂, --O--(CH₂)_(m)-aryl, NHC(═O)R₁ or halogeno, wherein m is 1 or 2;

Y is C₁₋₆ alkyl, C₁₋₆ alkenyl, aryl or arylalkyl;

n is 1unless X is B(OH)₂ and then n is zero;

R₁ is H, C₁₋₆ alkyl, aryl or arylalkyl;

P₂ is a bond, or a residue of Leu, Ala, Met, Ile, Val, Nva, Nle, Phe,Asp, Ser, Pro, His, cyclopentylglycine, cyclohexylglycine, tert-leucineor --HN--CH[CH₂ Si(C₁₋₆ alkyl)₂ (X)]C(═O)--;

P₃ is a bond or a residue of Val, Leu, Ile or Met;

P₄ is a bond or a residue of Val, Leu, Ile or Met;

K is hydrogen, a desamino group, formyl, acetyl, succinyl, benzoyl,t-butyloxycarbonyl, carbobenzyloxy, tosyl, dansyl, isovaleryl,methoxysuccinyl, 1-adamantanesulphonyl, 1-adamantaneacetyl,2-carboxybenzoyl, phenylacetyl, t-butylacetyl, bis[(1-naphthyl)methyl]acetyl, -A-R_(z) wherein ##STR1## R_(z) is an arylor an arylalkyl in which the aryl group contains 6, 10 or 12 carbons,the aryl group being suitably substituted by 1 to 3 members selectedindependently from the group consisting of fluoro, chloro, bromo, iodo,trifluoromethyl, hydroxy, alkyl containing from 1 to 6 carbons, alkoxycontaining from 1 to 6 carbons, carboxy, alkylcarbonylamino wherein thealkyl group contains 1 to 6 carbons, 5-tetrazolyl, and acylsulfonamido(i.e., acylaminosulfonyl and sulfonylaminocarbonyl) containing from 1 to15 carbons, provided that when the acylsulfonamido contains an aryl thearyl may be further substituted by a member selected from fluoro,chloro, bromo, iodo and nitro; and such other terminal amino protectinggroups which are functionally equivalent thereto, or ##STR2## wherein Zis N or CH, and

D is a group of the formulae ##STR3## (the wavy line being theattachment to the rest of the molecule, i.e., not to Z)

and wherein R' is hydrogen or a C₁₋₆ alkyl group, provided that X is notH, R is not benzyl, P₂ is not Val, P₃ is not a bond, P₄ is not a bondand K is not carbobenzyloxy, simultaneously. Theor preveunds are usefulfor inhibiting or preventing-the amyloid protein deposits in brain whichare associated with Alzheimer's disease and Down's Syndrome.

A compound of the formula IB or the hydrate, stereoisomer, isostere orpharmaceutically acceptable salt thereof (which are believed to becompounds of formula IA which have not been previously disclosed):

    K.sup.a --P.sub.4.sup.a --P.sub.3.sup.a --P.sub.2.sup.a --NH--C(R.sup.a)--[C(═O)].sub.n --X.sup.a (SEQ ID NO:2)IB

wherein

X^(a) is H, CHF₂, CF₃, CF₂ F₃, CF₂ CH₂ NHC(═O)R₁ ^(a), CHFCH₂ NHC(═O)R₁^(a), CF₂ C(═O)W^(a), C(═O)NHR₁ ^(a), B(OH)₂ or C(═O)R₁ ^(a),

wherein W^(a) is NHCH₂ Si(C₁₋₆ alkyl)₂ (y^(a)), NHR₁ ^(a) or R₁ ^(a) ;

provided that:

when X^(a) is H, then R^(a) is CH₂ Si(C₁₋₆.sbsb.-- alkyl)₂ (C₁₋₆ alkenylor aryl), CH₂ CH(CF₃)₂, CH₂ CH(CH₃)(CF₃), benzyl substituted withNHC(═O)R₁ ^(a), or NHC(═NH)NH₂ ;

when X^(a) is CF₃, CHF₂, or C(═O)NHR₁ ^(a) then R^(a) is not C₁₋₁₀alkyl, benzyl, CH₂ OH, CH(OH)CH₃, or benzyl substituted with one hydroxymoiety;

when X^(a) is CF₂ CH₂ NHC(═O)R₁ ^(a) then R^(a) is not C₁₋₁₀ alkyl,benzyl, (CH₂)m-napthyl or benzyl substituted with one hydroxy moiety;

when X^(a) is CF₂ C(═O)NH-benzyl then R^(a) is not benzyl,t-butyl-methyl or (CH₂)_(m) -napthyl;

when X^(a) is CF₂ C(═O)NHR₁ ^(a) then R^(a) is not CH₂ Si(CH₃)₂ (Y),C₁₋₁₀ alkyl, benzyl, CH₂ OH, CH(OH)CH₃, or benzyl substituted with onehydroxy moiety, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ alkyoxyalkyl, benzyloxy or--O--(CH₂)_(m) -phenyl;

when X^(a) is C(═O)R₁ ^(a) then R^(a) is not C₁₋₁₀ alkyl, benzyl, C₁₋₄alklylene-O-C₁₋₁₀ alkyl, or CH₂)_(m) -napthyl;

when X^(a) is CF₂ C(═O)phenethyl then R^(a) is not benzyl;

X^(a) is not C(═O)H when R^(a) is CH₂ Si(C₁₋₆ alkyl)₂ (C₁₋₆ alkenyl oraryl), CH₂ CH(CF₃)₂, CH₂ CH(CH₃)(CF₃), or benzyl substituted withNHC(═O)R₁ ^(a) or NHC(═NH)NH₂,

R^(a) is C₁₋₁₀ alkyl, benzyl, CH₂ Si(C₁₋₆ alkyl)₂ (Y^(a)), C₁₋₄alkylene-O--R₁ ^(a), CH₂ CH(CF₃)₂, CH₂ CH(CH₃)(CF₃), (CH₂)_(m)-naphthyl, or a substituted benzyl, the substitution being 1, 2 or 3substituents independently selected from the group consisting of C₁₋₆alkyl, C₁₋₆ alkoxy, C₁₋₆ alkoxyalkyl, benzyloxy, hydroxy, NHC(═NH)NH₂,NR₁ ^(a) H, NO₂, --O--(CH₂)_(m) -aryl, NHC(═O)R₁ ^(a) or halogeno,wherein m is 1 or 2;

Y^(a) is C₁₋₆ alkyl, C₁₋₆ alkenyl, aryl or arylalkyl;

n is 1 unless X^(a) is B(OH)₂ and then n is zero;

R₁ ^(a) is hydrogen, C₁₋₆ alkyl, aryl or arylalkyl,

P₂ ^(a) is a bond, --HN--CH[CH₂ Si(C₁₋₆ alkyl)₂ (Y^(a))]C(═O)-- or aresidue of Leu, Ala, Met, Ile, Val, Nva, Nle, Phe, Asp, Ser, Pro, His,cyclopentyl-glycine, cyclohexylglycine, or tert-leucine;

P₃ ^(a) is a bond or a residue of Val, Leu, Ile or Met;

P₄ ^(a) is a bond or a residue of Val, Leu, Ile or Met;

K^(a) is hydrogen, a desamino group, formyl, acetyl, succinyl, benzoyl,t-butyloxycarbonyl, carbobenzyloxy, tosyl, dansyl, isovaleryl,methoxysuccinyl, 1-adamantanesulphonyl, 1-adamantaneacetyl,2-carboxybenzoyl, phenylacetyl, t-butylacetyl, bis[(1-naphthyl)methyl]acetyl, -A^(a) -R_(z) ^(a) wherein ##STR4## R_(z)^(a) is an aryl or arylalkyl group in which the aryl group contains 6,10 or 12 carbons suitably substituted by 1 to 3 members selectedindependently from the group consisting of fluoro, chloro, bromo, iodo,trifluoromethyl, hydroxy, alkyl containing from 1 to 6 carbons, alkoxycontaining from 1 to 6 carbons, carboxy, alkylcarbonylamino wherein thealkyl group contains 1 to 6 carbons, 5-tetrazolyl, and acylsulfonamido(i.e., acylaminosulfonyl and sulfonylaminocarbonyl) containing from 1 to15 carbons, provided that when the acylsulfonamido contains an aryl thearyl may be further substituted by a member selected from fluoro,chloro, bromo, iodo and nitro; and such other terminal amino protectinggroups which are functionally equivalent thereto, or

wherein ##STR5## Z^(a) is N or CH, and D^(a) is a group of the formulae##STR6## and wherein R'^(a) is hydrogen or a C₁₋₆ alkyl group.

DETAILED DESCRIPTION OF THE INVENTION

A C₁₋₆ or C₁₋₁₀ alkyl group means straight, branched, cyclic alkylgroups or combinations thereof, for example, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, sec-pentyl,cyclopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl andcyclopentylmethyl. Likewise, C₁₆ alkylene and C₁₋₄ alkylene meanrespectively a one to six and a one to four carbon bivalent radicalwhich can be straight- or branched-chained. C₁₋₆ alkenyl has one to sixcarbons which are straight- or branched-chained with one or more doublebonds. All of the C₁₋₁₀ moieties are preferably C₁₋₆ moieties and morepreferably C₁₋₄ moieties. All of the C₁₋₆ moieties are preferably C₁₋₄moieties and more preferably C₁₋₂ moieties.

The compounds of formula I having aspartic or glutamic acid moieties maybe in free form or a salt form, e.g., acid addition or anionic salt.Such a compound may be converted into its salt or base form in anart-known manner, one from another. Preferred salts aretrifluoroacetate, hydrochloride, sodium, potassium or ammonium salts,although the scope is extended to include all of the salts known to beused in the art of peptide chemistry.

The term "stereoisomer" is a general term for all isomers of individualmolecules that differ only in the orientation of their atoms in space.It includes mirror image isomers (enantiomers), geometric (cis/trans)isomers, and isomers of compounds with more than one chiral center thatare not mirror images of one another (diastereo-isomers). Foramino-acids, the designations D/L or R/S can be used as described inIUPAC-IUB Joint Commission on Biochemical Nomenclature, Eur. J. Biochem.138: 9-37 (1984). The natural amino acids, with the exception ofglycine, contain a chiral carbon atom. Unless otherwise specificallyindicated, the preferred compounds are the optically active amino acidsof the L-configuration; however, applicants contemplate that the aminoacids of the formula I compounds can be of either the D- orL-configurations or can be mixtures of the D- and L-isomers, includingracemic mixtures. The recognized abbreviations for the α-amino acids areset forth in Table I.

As used herein "Alzheimer's Disease" also means senile dementia of theAlzheimer's type.

"Hydrate" means that the ketone of the compounds of this invention mayexist as a di-hydroxymethylene group. The compounds of the presentinvention are expected to be in the hydrated form under normalphysiological conditions.

"Desamino group" means an α-amino acid without the amino group attachedthereto. Preferred desamino groups are represented by the alpha aminoacids Val, Phe, Ala, Asp, Ser, and His, without their respectiveterminal amino groups.

"Isostere" means the normal peptide bond between attached amino acids(--C(O)NH--) is in a modified form of --CH₂ NH-- (reduced), C(O)N(CH₃)(N-methylamide), --COCH₂ -- (keto), --CH₂ (OH)CH₂ -- (hydroxy),--CH(NH₂)CH₂ -- (amino), --CH₂ CH₂ -- (hydrocarbon), or is inverted(--HN(C═O)--). Isostere as used herein also means an inversion of theC(═O)NH bond between an amino acid and the carbonyl moiety of theblocking group K. For example, Example 21 has two isostere groups: oneisostere is between the blocking group (K═[C₆ H₅ --(CH₂)₂ --C(═O)]-- andthe amino moiety of the valine residue, and the second isostere isbetween the valine residue and the phenylalanine aldehyde residue.Preferably the isostere only applies to the K-P₄ -P₃ -P₂ -moiety orportions thereof. Most preferably the compounds of the present inventionare not in isosteric forms.

"Aryl" means monocyclic or bicyclic carbocyclic ring systems having oneor more aromatic rings including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl and the like. Aryl groups can be substitutedor unsubstituted with one, two or three substituents independentlyselected from C₁₋₆ alkyl, haloalkyl, alkoxy, thioalkoxy,aminoalkylamino, dialkylamino, hydroxy, halo, mercapto, nitro,carboxaldehyde, carboxy, carboalkoxy and carboxamide. The above alkyl-and alkoxy compounds contain 1 to 6 carbons. Likewise, "arylalkyl" meansa C₁₋₆ alkylene, straight or branched chain, appended to an aryl asdefined herein, for example, benzyl.

"Substituted benzyl" means that a benzyl group is substituted at thephenyl moiety thereof at available carbon atoms, i.e., meta, orthoand/or para positions, having from one to three substituents. Preferablythere is only one substituent, and more preferably that substituent isat the para position.

Each α-amino acid has a characteristic "R-group", the R-group being theside chain, or residue, attached to the α-carbon atom of the α-aminoacid. For example, the R-group side chain for glycine is hydrogen, foralanine it is methyl, for valine it is isopropyl. For the specificR-groups or side chains of the α-amino acids see A. L. Lehninger's texton Biochemistry.

"(CH₂)_(m) -naphthyl" is a straight- or branched-chain alkylene attachedto naphthyl at the 1- or 2-position thereof.

The natural amino acids, with the exception of glycine, contain a chiralcarbon atom. Unless otherwise specifically indicated, the preferredcompounds are the optically active amino acids of the L-configuration;however, applicants contemplate that the amino acids of the formula Icompounds can be of either the D- or L-configurations or can be mixturesof the D- and L-isomers, including racemic mixtures. The recognizedabbreviations for the α-amino acids are set forth in Table I.

                  TABLE I                                                         ______________________________________                                        AMINO ACID     SYMBOL                                                         ______________________________________                                        Alanine        Ala                                                              Glycine Gly                                                                   Isoleucine Ile                                                                Leucine Leu                                                                   Lysine Lys                                                                    Serine Ser                                                                    Arginine Arg                                                                  Threonine Thr                                                                 Asparagine Asn                                                                Valine Val                                                                    Norvaline Nva                                                                 Norleucine Nle                                                                Glutamic acid Glu                                                             Cysteine Cys                                                                  Histidine His                                                               ______________________________________                                    

Note that even though formula IB has an "a" superscript to all of itsvariables, the only difference between formulae IA and IB is at X andX^(a) in the provisos. The following schemes are directed to variableswhich do not have an "a" superscript but are used to describe thesynthesis for both formulae IA and IB.

In general, the compounds of formula I may be prepared using standardchemical reaction analogously known in the art and as depicted in SchemeA. ##STR7##

Scheme A provides a general synthetic scheme for preparing the compoundsof formula I.

The P₂, P₃ and K-P₄ groups can be linked to the free amino group of theamino acid derivative of structure (1). The P₂, P₃ and K-P₄ can belinked to the unprotected, free amino compound by well known peptidecoupling techniques.

Generally, peptides are elongated by deprotecting the α-amine of theC-terminal residue and coupling the next suitably protected amino acidthrough a peptide linkage using the methods described. This deprotectionand coupling procedure is repeated until the desired sequence isobtained. This coupling can be performed with the constituent aminoacids in stepwise fashion, as depicted in Scheme A, or by condensationof fragments (two to several amino acids), or combination of bothprocesses, or by solid phase peptide synthesis according to the methodoriginally described by Merrifield, J. Am. Chem. Soc., 1963, 85,2149-2154, the disclosure of which is hereby incorporated by reference.When a solid phase synthetic approach is employed, the C-terminalcarboxylic acid is attached to an insoluble carrier (usuallypolystyrene). These insoluble carriers contain a group which will reactwith the aldehyde group to form a bond which is stable to the elongationconditions but readily cleaved later. Examples of which are chloro- orbromomethyl resin, hydroxymethyl resin, and aminomethyl resin. Many ofthese resins are commercially available with the desired C-terminalamino acid already incorporated. For compounds of formula I wherein X isH, a linker compound may also be used in the reaction of Scheme A tolink a resin to the aldehyde functionality of the amino acid derivativeof structure (1) wherein X is H. Examples of suitable linker compoundsare ##STR8##

Alternatively, compounds of the invention can be synthesized usingautomated peptide synthesizing equipment. In addition to the foregoing,peptide synthesis are described in Stewart and Young, "Solid PhasePeptide Synthesis", 2nd ed., Pierce Chemical Co., Rockford, Ill. (1984);Gross, Meienhofer, Udenfriend, Eds., "The Peptides: Analysis, Synthesis,Biology", Vol 1, 2, 3, 5 and 9, Academic Press, New York, 1980-1987;Bodanszky, "Peptide Chemistry: A Practical Textbook", Springer-Verlag,New York (1988); and Bodanszky, et al. "The Practice of PeptideSynthesis" Springer-Verlag, New York (1984), the disclosures of whichare hereby incorporated by reference.

Coupling between two amino acids, an amino acid and a peptide, or twopeptide fragments can be carried out using standard coupling proceduressuch as the azide method, mixed carbonic acid anhydride (isobutylchloroformate) method, carbodiimide (dicyclohexylcarbodiimide,diisopropylcarbodiimide, or water-soluble carbodiimide) method, activeester (p-nitrophenyl ester, N-hydroxy-succinic imido ester) method,Woodward reagent K method, carbonyldiimidazole method, phosphorusreagents such as BOP-Cl, or oxidation-reduction methods. Some of thesemethods (especially the carbodiimide method) can be enhanced by adding1-hydroxybenzotriazole. These coupling reactions can be performed ineither solution (liquid phase) or solid phase.

The functional groups of the constituent amino acids must be protectedduring the coupling reactions to avoid formation of undesired bonds. Theprotecting groups that can be used are listed in Greene, "ProtectiveGroups in Organic Chemistry", John Wiley & Sons, New York (1981) and"The Peptides: Analysis, Synthesis, Biology", Vol. 3, Academic Press,New York (1981), the disclosure of which is hereby incorporated byreference.

The α-carboxyl group of the C-terminal residue is usually protected byan ester that can be cleaved to give the carboxylic acid. Protectinggroups which can be used include: 1) alkyl esters such as methyl andt-butyl, 2) aryl esters such as benzyl and substituted benzyl, or 3)esters which can be cleaved by mild base treatment or mild reductivemeans such as trichloroethyl and phenacyl esters.

The α-amino group of each amino acid must be protected. Any protectinggroup known in the art can be used. Examples of which include: 1) acyltypes such as formyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl;2) aromatic carbamate types such as benzyloxycarbonyl (Cbz or Z) andsubstituted benzyloxycarbonyls, 1-(p-biphenyl)-1-methylethoxy-carbonyl,and 9-fluorenylmethyloxycarbonyl (Fmoc); 3) aliphatic carbamate typessuch as tertbutyloxycarbonyl (Boc), ethoxycarbonyl,diisopropylmethoxycarbonyl, and allyloxycarbonyl; 4) cyclic alkylcarbamate types such as cyclopentyloxycarbonyl andadamantyloxycarbonyl;; 5) alkyl types such as triphenylmethyl andbenzyl; 6) trialkylsilane such as trimethylsilane; and 7) thiolcontaining types such as phenylthio-carbonyl and dithiasuccinoyl. Thepreferred α-amino protecting group is either Boc or Fmoc, preferablyFmoc. Many amino acid derivatives suitably protected for peptidesynthesis are commercially available.

The α-amino protecting group is cleaved prior to the coupling of thenext amino acid. When the Boc group is used, the methods of choice aretrifluoroacetic acid, neat or in dichloromethane, or HCl in dioxane. Theresulting ammonium salt is then neutralized either prior to the couplingor insitu with basic solutions such as aqueous buffers, or tertiaryamines in dichloromethane or dimethylformamide. When the Fmoc group isused, the reagents of choice are piperidine or substituted piperidine indimethylformamide, but any secondary amine or aqueous basic solutionscan be used. The deprotection is carried out at a temperature between 0°C. and room temperature.

Any of the amino acid bearing side chain functionalities must beprotected during the preparation of the peptide using any of theabove-described groups. Those skilled in the art will appreciate thatthe selection and use of appropriate protecting groups for these sidechain functionalities depends upon the amino acid and presence of otherprotecting groups in the peptide. The selection of such protectinggroups is important in that it must not be removed during thedeprotection and coupling of the α-amino group.

For example, when Boc is used as the a-amino protecting group, thefollowing side chain protecting groups are suitable: p-toluenesulfonyl(tosyl) moieties can be used to protect the amino side chains of aminoacids such as Lys and Arg; p-methylbenzyl, acetamidomethyl, benzyl(Bzl), or t-butylsulfonyl moieties can be used to protect the sulfidecontaining side chains of amino acids such as cysteine; and benzyl (Bzl)ether can be used to protect the hydroxy containing side chains of aminoacids such as Ser or Thr.

When Fmoc is chosen for the a-amine protection usually tert-butyl basedprotecting groups are acceptable. For instance, Boc can be used forlysine, tert-butyl ether for serine and threonine and tert-butyl esterfor glutamic acid.

Once the elongation of the peptide is completed all of the protectinggroups are removed. When a solution phase synthesis is used, theprotecting groups are removed in whatever manner is dictated by thechoice of protecting groups. These procedures are well known to thoseskilled in the art.

When a solid phase synthesis is used, the peptide is cleaved from theresin usually simultaneously with the protecting group removal. When theBoc protection scheme is used in the synthesis, treatment with anhydrousHF containing additives such as dimethyl sulfide, anisole, thioanisole,or p-cresol at 0° C. is the preferred method for cleaving the peptidefrom the resin. The cleavage of the peptide can also be accomplished byother acidic reagents such as trifluoromethanesulfonicacid/trifluoroacetic acid mixtures. If the Fmoc protection scheme isused the N-terminal Fmoc group is cleaved with reagents describedearlier. The other protecting groups and the peptide are cleaved fromthe resin using a solution of trifluoroacetic acid and various additivessuch as anisole, etc.

For those compounds of formula I wherein X is H, the peptide compound offormula I may be cleaved from the linker compound and resin with aqueousacid/formaldehyde.

Alternatively, the compounds of formula I may be prepared using standardchemical reactions analogously known in the art and as depicted inScheme B. ##STR9## Scheme B provides an alternative general syntheticscheme for preparing the compounds of formula I.

The P₂, P₃ and K-P₄ groups can be linked to the free amino group of theamino alcohol derivative of structure (2) as described previously inScheme A to give the peptido alcohol of structure (3).

The alcohol functionality of the peptido alcohol of structure (3) isthen oxidized by techniques and procedures well known and appreciated byone of ordinary skill in the art, such as a Swern Oxidation using oxalylchloride and dimethylsulfoxide, to give the compounds of formula I.

Starting materials for use in Schemes A and B are readily available toone of ordinary skill in the art. For example, amino acids P₂, P₃ andK-P₄ wherein K is hydrogen are commercially available and the linkercompound of structure (L1) is described in J. Am. Chem. Soc., 114,3157-59 (1992). In addition, substituted amino acids K-P₄ wherein K isacetyl, succinyl, benzoyl, t-butyloxycarbonyl, carbobenzyloxy, tosyl,dansyl, isovaleryl, methoxysuccinyl, 1-adamantanesulphonyl,1-adamantaneacetyl, 2-carboxbenzoyl, phenylacetyl, t-butylacetyl, bis[(1-naphthyl)methyl]acetyl or -A-R_(z) wherein ##STR10##

Rz is an aryl group containing 6, 10 or 12 carbons suitably substitutedby 1 to 3 members selected independently from the group consisting offluoro, chloro, bromo, iodo, trifluoromethyl, hydroxy, alkyl containingfrom 1 to 6 carbons, alkoxy containing from 1 to 6 carbons, carboxy,alkylcarbonylamino wherein the alkyl group contains 1 to 6 carbons,5-tetrazolyl, and acylsulfonamido (i.e., acylaminosulfonyl andsulfonylaminocarbonyl) containing from 1 to 15 carbons, provided thatwhen the acylsulfonamido contains an aryl the aryl may be furthersubstituted by a member selected from fluoro, chloro, bromo, iodo andnitro; and such other terminal amino protecting groups which arefunctionally equivalent thereto are described in European PatentApplication No. 0363284, Apr. 11, 1990.

Starting amino compounds of formula (1) are readily available to one ofordinary skill in the art. For example, certain protected aminocompounds of Formula IA wherein:

X is H and R is benzyl is described in European Patent Application No.0363284 and WO84/00365;

X is H and R is CH₂ Si(CH₃)₃ is described in European Patent ApplicationNo. 0363284 and described herein in Examples 12. As described in Example13 and 14, substituents on the silyl can be different;

X is H and R is substituted benzyl is described in Patent ApplicationPCT/US91/09741;

X is CF₃ and CHF₂, and R is benzyl or benzyl substituted with NHC(NH)NH₂are described in European Patent Application No. 0195212 with apublication date of Sep. 24, 1986, inventors Michel Jung et al.;

X is CF₂ CH₂ NHC(═O)R1 and R is benzyl is described as an intermediatein European Patent Application OPI No. 0275101, filed Jan. 14, 1988,inventors Daniel Schirlin et al.; the monofluoro derivative CFHCH₂NHC(═O)R1 can be synthesized by similar methods using bromo-fluoroaceticacid, ethyl ester in place of bromo-difluoroacetic acid, ethyl ester;

X is CF₂ C(O)W wherein W is NHCH₂ Si(alkyl)₃ and R is benzyl, CH₂Si(CH₃)₃ or substituted benzyl are described in Patent Application No.PCT/US91/09741, inventors Daniel Schirlin et al., filed Dec. 20, 1991,and when R is (CH₂)_(m) -naphthyl similar methods may be used withstarting materials well known in the art;

X is CF₂ C(O)W wherein W is NHR1 or R1 and R is benzyl, CH₂ Si(CH₃)₃ orsubstituted benzyl are described in Patent Application No.PCT/US91/09741, inventors Daniel Schirlin et al., filed Dec. 20, 1991,and when R is (CH₂)_(m) -naphthyl similar methods may be used withstarting materials well known in the art;

X is C(O)R1 and R is benzyl, CH₂ Si(CH₃)₃, (CH₂)_(m) -naphthyl orsubstituted benzyl are described in U.S. Pat. No. 4,820,691, filed Apr.11, 1989; and

The linker compound trans-4-(aminomethyl-cyclohexane)carboxylic acid,benzyl ester used in the synthesis of compound of formula I wherein X isH is prepared from the corresponding acid as described in J. Am. Chem.Soc. 1992, 114, 3156-3157.

All of the foregoing cites are hereby incorporated herein by reference.

In addition, other starting materials for use in Schemes A and B may beprepared by the following synthetic procedures which are well known andappreciated by one of ordinary skill in the art.

Substituted amino acids K-P₄ of structure wherein K is ##STR11## whereinZ is N or CH, and

D is a group of the formulae ##STR12## wherein R' is hydrogen or a C₁₋₆alkyl group are prepared using standard chemical reactions analogouslyknown in the art.

The procedure for preparing the substituted amino acids K-P₄ wherein Kis ##STR13## wherein D is a --C(═O)-- is outlined in Scheme B wherein P₄and Z are as previously defined or are the functional equivalents ofthese groups. ##STR14##

Specifically the amino acids K-P₄ wherein K is ##STR15## wherein D is a--C(═O)--are prepared by coupling of the amino acid K-P₄ wherein K ishydrogen with acid chloride of structure (4) in the presence of from oneto four molar equivalents of a suitable amine which can act as ahydrogen halide acceptor. Suitable amines for use as hydrogen halideacceptors are tertiary organic amines such as tri-(lower alkyl)amines,for example, triethylamine, or aromatic amines such as picolines,collidines, and pyridine. When pyridines, picolines, or collidines areemployed, they can be used in high excess and act therefore also as thereaction solvent. Particularly suitable for the reaction isN-methylmorpholine ("NMM"). The coupling reaction can be performed byadding an excess, such as from 1-5, preferably about a 4-fold molarexcess of the amine and then the acid chloride of structure (4), to asolution of the amino acid K-P₄ wherein K is hydrogen. The solvent canbe any suitable solvent, for example, petroleum ethers, a chlorinatedhydrocarbon such as carbon tetrachloride, ethylene chloride, methylenechloride, or chloroform; a chlorinated aromatic such as1,2,4-trichlorobenzene, or o-dichlorobenzene; carbon disulfide; anethereal solvent such as diethylether, tetrahydrofuran, or 1,4-dioxane,or an aromatic solvent such as benzene, toluene, or xylene. Methylenechloride is the preferred solvent for this coupling reaction. Thereaction is allowed to proceed for from about 15 minutes to about 6hours, depending on the reactants, the solvent, the concentrations, andother factors, such as the temperature which can be from about 0° C. toabout 60° C., conveniently at about room temperature, i.e. 25° C. Theamino acids K-P₄ wherein K is ##STR16## wherein D is a --C(═O)-- can beisolated from the reaction mixture by any appropriate techniques such asby chromatography on silica gel.

The substituted amino acids K-P₄ wherein K is ##STR17## wherein D isother then a --C(═O)--can be prepared analogously, merely bysubstituting the appropriate intermediate ##STR18## wherein D is otherthan a --C(═O)--and E is Cl or OH (the corresponding acid, acid chlorideor sulphonyl chloride) for the compound of structure (5) in Scheme C.

The acid chloride of structure (4) and the appropriate intermediate offormula ##STR19## wherein

B is other then a --C(═O)-- and E is Cl or OH (the corresponding acid,acid chloride or sulphonyl chloride) are commercially available or maybe readily prepared by techniques and procedures well known andappreciated by one of ordinary skill in the art.

For example, the appropriate intermediates of formula ##STR20## may beprepared as outlined in Scheme D wherein all substituents are aspreviously defined. ##STR21##

Scheme D provides a general synthetic procedure for reparing theappropriate intermediates of formula ##STR22## Z is as previouslydefined.

In step a, carboxylic acid functionality of the appropriate2,5-pyridinedicarboxylic acid, 2-methyl ester (6) (Nippon Kagaku Zasshi,1967, 88, 563) is converted to its acid chloride using techniques andprocedures well known and appreciated by one of ordinary skill in theart, such as thionyl chloride, to give the corresponding2,5-pyridinedicarboxylic acid, 2-methyl ester, 5-acid chloride (7)

In step b, the 2,5-pyridinedicarboxylic acid, 2-methyl ester, 5-acidchloride (7) is amidated with morpholine (8) by techniques andprocedures well known and appreciated by one of ordinary skill in theart to give the corresponding 2,5-pyridinedicarboxylic acid, 2-methylester, 3-morpholino amide (9).

In step c, the methyl ester functionality of 2,5-pyridinedicarboxylicacid, 2-methyl ester, 3-morpholino amide (9) is hydrolyzed by techniquesand procedures well known and appreciated by one of ordinary skill inthe art, with for example, lithium hydroxide in methanol, to give the2,5-pyridinedicarboxylic acid, 5-morpholino amide (10).

In addition, the appropriate intermediate of formula ##STR23## may beprepared as outlined in Scheme E wherein all substituents are aspreviously defined. ##STR24##

Scheme E provides a general synthetic procedure for preparing theappropriate intermediates of formula ##STR25## wherein

Z is as previously defined.

In step a, the free carboxylic acid functionality of2,5-pyridinedicarboxylic acid, 2-methyl ester (6) (Nippon Kagaku Zasshi,1967, 88, 563) is converted to its t-butyl ester using techniques andprocedures well known and appreciated by one of ordinary skill in theart, such as the t-butyl alcohol adduct of dicyclohexylcarbodiimide(Synthesis, 1979, 570), to give the corresponding2,5-pyridinedicarboxylic acid, 2-methyl ester, 5-t-butyl ester (11) .

For example, the 2,5-pyridinedicarboxylic acid, 2-methyl ester (6) isreacted with a molar excess of the t-butyl alcohol adduct ofdicyclohexylcarbodiimide in an appropriate organic solvent, such asmethylene chloride. The reaction is typically conducted at a temperaturerange of from 0° C. to room temperature and for a period of time rangingfrom 2-24 hours. The 2,5-pyridinedicarboxylic acid, 2-methyl ester,5-t-butyl ester (11) is isolated from the reaction zone by standardextractive methods as is known in the art. and may be purified bycrystallization.

In Step b, the methyl ester functionality of 2,5-pyridinedicarboxylicacid, 2-methyl ester, 5-t-butyl ester (11) is amidated with morpholine(8) to give the corresponding 2,5-pyridinedicarboxylic acid,2-morpholino amide, 5-t-butyl ester (12).

For example, the 2,5-pyridinedicarboxylic acid, 2-methyl ester,5-t-butyl ester (11) is contacted with molar excess of morpholine in anappropriate organic solvent, such as tetrahydrofuran. The reaction istypically conducted at a temperature range of from room temperature toreflux and for a period of time ranging from 5 hours to 3 days. The2,5-pyridinedicarboxylic acid, 2-morpholino amide, 5-t-butyl ester (12)is isolated from the reaction zone by standard extractive methods as isknown in the art. and may be purified by crystallization.

In step c, the t-butyl ester functionality of 2,5-pyridinedicarboxylicacid, 2-morpholino amide, 5-t-butyl ester (12) is hydrolyzed, with forexample, HCl in nitromethane, to give the corresponding,2,5-pyridine-dicarboxylic acid, 2-morpholino amide (13).

In general, the compounds of formula IA may be prepared using standardchemical reactions analogously known in the art. For example, asynthesis of the compounds of formula IA where X is H is depicted inScheme F. All the substituents, unless otherwise indicated, are aspreviously defined. The reagents and starting materials are readilyavailable to one of ordinary skill in the art. ##STR26##

The required starting material defined by compound (14) is readilyavailable either commercially or by applying known prior art principlesand techniques. The term "Pg" refers to a suitable protecting group asmore fully defined previously. Examples of such compounds are thesuitably protected amino acids serine, homoserine, threonine,allothreonine and the like. In addition, L-lysine can be transformedinto 2(S)-2-amino-6-hydroxyhexanoic acid following generally theprocedure described by Baldwin, J. E. et al., Tetrahedron, 44, 2633(1988), incorporated herein.

In Scheme F, Step A the protected amino acid (14) is transformed intothe amide (15). This amidation can be performed utilizing a couplingreaction as between two amino acids using the protected amino acid (14)and the N-alkyl O-alkylhydroxylamine. The standard coupling reaction canbe carried out using standard coupling procedures as describedpreviously for the coupling between two amino acids to provide the amide(15).

In Scheme F, Step B the amide (15) is deprotected under conditions wellknown in the art as described by T. H. Green, "Protective Groups inOrganic Synthesis", John Wiley and Sons, 1981, Chapter 7, to provide thedeprotected amide (16). For example, when "Pg" is a t-butyloxycarbonyl(Boc), the amide (15) is dissolved in a suitable solvent, such as ethylacetate treated with excess hydrogen chloride (gas) and stirred at about0° C. to 30° C. for about 30 minutes to 4 hours. The solvent is thenremoved under vacuum to provide the deprotected amide (16) as the HClsalt.

In Scheme F, Step C the deprotected amide (16) is elongated by couplingthe next suitably protected amino acid through a peptide linkage usingthe methods previously described in Scheme A, or by condensation offragments, or combination of both processes to provide the elongatedpeptide (17).

In Scheme F, Step D the elongated peptide (17) is reduced to provide thedesired aldehyde of formula IA.

For example, the elongated peptide (17) is dissolved in a suitableorganic solvent, such as tetrahydrofuran and cooled to 0° C. under anatmosphere of nitrogen. An excess of a suitable reducing agent is addedto the solution. Examples of suitable reducing agents are lithiumaluminum hydride, diisobutylaluminum hydrides,tri-tert-butyloxy-aluminum hydrides, sodium aluminum hydrides,diamino-aluminum hydrides and the like. The preferred reducing agent islithium aluminum hydride. The reaction is stirred for 20 minutes to 2hours at a temperature of about 0° C. to 20° C. The reaction is thenquenched and the product isolated by techniques well known in the art.For example, the reaction is quenched with 10% potassium hydrogensulfate followed by addition of 10% hydrochloric acid. The aqueousmixture is then extracted with a suitable organic solvent, such as ethylacetate. The organic extract is washed with water, dried over magnesiumsulfate, filtered and concentrated under vacuum to provide the aldehydeof formula IA.

The compounds of formula IA, wherein X is C(═O)NHR₁ can be preparedfollowing the procedure described in Scheme F. All the substituents,unless otherwise indicated, are previously defined. The reagents andstarting materials are readily available to one of ordinary skill in theart. ##STR27##

In Scheme G, Step A, the α-keto ester (18) is selectively hydrolyzed tothe α-keto acid (19) by treatment with a suitable base. [The α-ketoester (18) is readily prepared following generally the proceduredescribed by Angelastro, M. R. et al., J. Med. Chem., 33, 11 (1990).]

For example the appropriately substituted α-keto ester (18) is dissolvedin a suitable solvent mixture, such as methanol:water (50:50) andtreated with an equivalent of a suitable base, such as lithiumhydroxide. The reaction is stirred at a temperature of about 0° C. to30° C. for about 1 to 10 hours. The α-keto acid (19) is then isolated byextractive techniques well known in the art. For example, the reactionis diluted with a suitable organic solvent, such as ethyl acetate and anequal volume of water. The layers are separated. The aqueous layer isacidified with dilute hydrochloric acid and extracted with a suitableorganic solvent, such as ethyl acetate. The combined organic extractsare dried over anhydrous magnesium sulfate, filtered and concentratedunder vacuum to provide the α-keto acid (19).

In Scheme G, Step B the α-keto acid (19) is coupled with a primary amine(20) under conditions well known in the art to provide the desireda-keto amide (21).

For example, the appropriately substituted α-keto acid (19) is dissolvedin a suitable organic solvent, such as methylene chloride. The solutionis then treated with one equivalent of 1-hydroxybenzotriazole, oneequivalent of diisopropylethylamine and one equivalent of a primaryamine (20). An equivalent of dicyclohexylcarbodiimide is added and thereaction is stirred at a temperature of about 0° C. to 25° C. for about2 to 10 hours. The product is then isolated by techniques well known inthe art. For example, the reaction is diluted with ethyl acetate, rinsedwith cold 0.5 N hydrochloric acid, saturated sodium bicarbonate, driedover anhydrous magnesium sulfate, filtered and concentrated under vacuumto provide the α-keto amide (21).

In Scheme G, Step C the α-keto amide (21) is deprotected underconditions well known in the art as described by T. H. Green,"Protective Groups in Organic Synthesis", John Wiley and Sons, 1981,Chapter 7, to provide the deprotected a-keto amide (22). For example,when "Pg" is a t-butyloxycarbonyl (Boc), the α-keto amide (21) isdissolved in a suitable solvent, such as ethyl acetate treated withexcess hydrogen chloride (gas) and stirred at about 0° C. to 30° C. forabout 30 minutes to 4 hours. The solvent is then removed under vacuum toprovide the deprotected a-keto amide (22) as the HCl salt.

The deprotected α-keto amide (22) is then subjected to the reactionconditions described in Scheme A to provide the compounds of formula IAwherein X is C(═O)NHR₁.

In formula IA, wherein X is B(OH)₂, the following general scheme may befollowed (other starting materials where R is other than benzyl may beused as is well knwon in the art): ##STR28##

In Scheme H, the boronic acid derivative (23) is protected by aprotecting group (Pg₁), which can be cyclic (e.g., Pg₁ =pinane) or twoseparate protecting groups.

In Step A, a homologation occurs and a leaving group (Lg) such aschloride is introduced. Then, in Step B, the boronate (24) is aminated.The amine (25) is preferably protected with any two protecting groups(Pg₂ and Pg₃). For example, see descriptions in J. Am. Chem. Soc. 1981,103: 5241-5242. In Step C, the amine may be deprotected as previouslydescribed herein or as well known in the art. Coupling in Step C mayoccur as previously described in Scheme A. The boronate moiety of thecoupled compound (27) may then be deprotected by well known means (seeJ. Am. Chem. Soc., 1981, 103: 5241-5242 for an example of the foregoingreactions).

In order to incorporate a substituent of the following type: NR₁ H, thestarting material in Scheme I should be in a protected form as follows:##STR29##

When the substituent on the benzyl (R side chain) is NO₂, the followingscheme is preferred ##STR30##

In step a, the --NO₂ phenylalanine derivative is converted to an esterusing techniques and procedures well known and appreciated by one ofordinary skill in the art, such as methanol in presence ofdicyclohexylcarbodiimide and 4-dimethyl aminopyridine.

In step b, the ester (29) is deprotected under conditions well known inthe art as described by T. H. Green, "Protective Groups in OrganicSynthesis", John Wiley and Sons, 1981, Chapter 7, to provide thedeprotected ester (30).

In step c, the deprotected ester (30) is elongated by coupling the nextsuitably protected amino acid through a peptide linkage using themethods previously described in Scheme I, or by condensation offragments, or combination of both processes to provide the elongatedpeptide (31).

In step d, the elongated peptide (31) is reduced to aldehyde (32) usingtechniques and procedures well known and appreciated by one of ordinaryskill in the art, such as diisobutylaluminum hydride (Dibal) in atoluene/diethyl ether mixture at low temperature (--78° C. to -50° C.).

Compounds of formula IA with X being CHF₂ or CF₃, can be preparedaccording to scheme J.

For those compounds wherein X is either --CF₂ H or --CF₃, intermediatesfor the application of the standard peptide coupling techniques arecompounds of formula IIa-b ##STR31##

wherein X' is --CF₃ or --CF₂ H, and R is as previously defined informula IA. Similarly, designations P₁, P₂, P₃, P₄, and K shown in theforegoing schemes are as defined in formula IA, except that anysubgeneric or other modifications thereof (as in X) are highlighted bythe use of a primed symbol with a specific designation for such modifiedsymbol. The preparation and application of these compounds are depictedin scheme J. ##STR32## wherein R₆ is alkyl, phenyl or other equivalentmoiety, and X' is --CF₂ H or --CF₃,. H⊖A'⊖ means an acid.

In general the formation of the substituted azlactones (34) is effectedfrom the N-protected amino acids (33) by standard reaction conditionswherein the amino acid derivative (33) is heated in the presence of anacid anhydride. The so-produced azlactone (34) is reacted with a di- ortrifluoroacetic acid anhydride or acid halide to give a fluorinatedintermediate which (with or without isolation) is treated with anhydrousoxalic acid to produce the N-protected fluorinated ketone (35) whereuponthe ketone is chemically reduced to its alcoholic amide (36). The amide(36) is cleaved under standard acidic conditions to yield its amide acidsalt [e.g., its hydrochloride (37)]. After neutralization, the alcohols(IIa) may be coupled to KP₄ P₃ P₂ OH using standard peptide chemistrytechniques to produce compounds (38) which are subjected to the Swernoxidation procedure to obtain the desired product (39a) and (39b) (theketone or hydrate respectively). Alternatively, the alcohols (IIa) maybe oxidized to the ketones (IIb) which are coupled to KP₄ P₃ P₂ OHaccording to standard peptide chemistry techniques. When employing thisalternative route, the amino moiety is first protected with a Bocprotecting group, the OH function oxidized to its ketone via Swernoxidation procedures, and then the Boc protecting group removed and theresulting compounds (IIb) are the coupled to KP₄ P₃ P₂ OH.

Scheme J is also applicable for the preparation of compounds of formulaIA wherein X is CF₂ CF₃, the substituted azlactones (34) being heated inthe presence of penta-fluoropropanoic acid anhydride or acid halide.

An alternate route for the preparation of compounds of formula IAwherein X=CF₂ CF₃, is shown in scheme K. ##STR33##

In step a the protected amino acid (40) is transformed into thehydroxamate (41). This amidation can be performed utilizing a couplingreaction as between two amino acids using the protected amino acid (40)and the N-alkyl O-alkylhydroxylamine. The standard coupling reaction canbe carried out using standard coupling procedures as describedpreviously for the coupling between two amino acids to provide thehydroxamate (41).

In step b, the protected hydroxamate (41) is transformed into theprotected pentafluoroketone (43) [or (44)]. This reaction can beperformed utilizing a coupling reaction of the type described in thefollowing reference M. R. Angelastro, J. P Burkhart, P. Bey, N. P. Peet,Tetrahedron Letters, 33 (1992), 3265-3268.

In step c, the hydroxamate (41) is deprotected under conditions wellknown in the art as described by T. H. Green "Protection Groups inOrganic Synthesis", John Wiley and Sons, 1981, Chapter 7, to provide thedeprotected hydroxamate (41). The deprotected hydroxamate (41) iselongated by coupling the next suitably protected amino acid through apeptide linkage using the methods previously described in Scheme K, orby condensation of fragments, or combination of both processes toprovide the elongated peptide (42).

In step d, the ketone (43) is deprotected under conditions as previouslydescribed. The deprotected ketone (43) is elongated by coupling the nextsuitably protected amino acid through a peptide linkage using themethods previously described in Scheme K, or by condensation offragments, or combination of both processes to provide the elongatedketone (43).

For the preparation of compounds of formula IA wherein X is CF₂ CH₂NHCOR₁ the following schemes may be used. ##STR34##

In effecting the steps of scheme L it is preferred to start with thealdehyde (45) wherein the protecting group is a carbamate preferablywherein Pg is benzyloxycarbonyl (CBZ). This so-protected aldehyde issubjected to a condensation reaction with an ester ofbromodifluoroacetic acid, preferably the ethyl ester in the presence ofzinc. Preferably the reaction is conducted in an anhydrous aproticsolvent, e.g., tetrahydrofuran, ether, dimethoxy-ethane and the likeunder a nitrogen atmosphere. The reaction mixture is gently heated underreflux conditions, preferably to about 60° C. for about 1-12 hours. Theester (46) is converted to its primary amide ((47) by treatment withliquid ammonia under anhydrous conditions, preferably using suchsolvents as anhydrous diethyl ether. The amidation is initiated at -78°C. and following saturation with ammonia the reaction mixture is slowlyallowed to rise to room temperature. The so-formed amide is chemicallyreduced to form the free amine. This chemical reduction is easilyeffected by reacting the amide with a diborane, preferably as adiborane/dimethylsulfide complex, under a nitrogen atmosphere in ananhydrous aprotic solvent (e.g., THF) under reflux conditions. Thereduction yields the desired amine, in the form of an acid (e.g., HCl)salt which, by pH adjustment, yields the free amine which may besuitably protected with an N-protecting group, e.g., P'g is t-butoxycarbonyl using the standard reaction conditions (e.g., (BOC)₂ O, THF atroom temperature) for protection the amine. Alternatively the free aminemay be subjected to reaction conditions designed to build the desireda-amino acid or peptide moiety on the P' side of the difluoro-methylenemoiety.

Having obtained the intermediates of formula (48) standard α-amino acidor peptide coupling procedures may be conducted to prepare theindividual compounds of formula IA. In practice it is more convenient toeffect coupling on the P' side of the difluoromethylene moiety.

For compounds of formula IA wherein X is CF₂ CH₂ NHCOR₁, the followingscheme M should be used. ##STR35##

The oxidation step may be effected via the well known Swern oxidationprocedure, or with a modified Jones reaction using pyridiniumdichromate, or a chromic anhydride pyridinium complex, or with1,1,1-triacetoxy-2,1-benzoxiodol (Dess-Martin reagent).

For the preparation of compounds of formula IA wherein X is CHFCH₂NHCOR₁, scheme L could be used, the first step being a condensationreaction between aldehyde (45) and an ester of bromofluoroacetic acid,preferably ethyl ester in the presence of zinc. Preferably, the reactionis conducted in an anhydrous aprotic solvent (THF, ether,dimethoxyethane) and under nitrogen. Conditions are as described forscheme L.

For the preparation of compounds of formula IA wherein X is CF₂ (C═O)Wand W is NHCH₂ Si(C₁₋₆ alkyl)₂ (B) or NHR₁, scheme N may be used.##STR36##

Step a is similar to scheme L step a, and applicable to all side chainsof the present invention. Ester (54) in scheme N is converted to thesecondary amide (55) by treatment with the corresponding primary amines(58) or (59) under anhydrous conditions, preferably using such solventsas THF. The amidation is initiated at 0° C. or at room temperature andthe reaction mixture might be heated to reflux for completion of thereaction.

In step c, the so-formed amide (55) is deprotected under conditionssimilar to the one described in scheme F, step b. The deprotected amideis elongated by coupling the next suitably protected amine and through apeptide linkage using the methods previously described in scheme A or bycondensation of fragments, or by combination of both processes toprovide the elongated peptide (56).

In step d the alcohol functionality of the alcohol(56) is then oxidizedby techniques and procedures well known and appreciated of one ordinaryskill in the art, such as Swern oxidation using oxalyl chloride anddimethyl-sulfoxide, to give the compounds of formula (58).

For compounds of formula IA wherein X is CF₂ C(═O)W and W═R₁, thefollowing scheme may be used. ##STR37##

In step a, chlorodifluoromethy ketones (61) are preferably prepared byaddition of an organometallic derivative of type MR₁ (derived from R₁halogeno) (preferably Li or Mg) in an inert solvent under anhydrousconditions (e.g., THF) at low temperature (preferably 0° C.) tochlorodifluorodimethylhydroxamate (60).

In step a', chlorodifluoromethy ketones (61) are preferably prepared byaddition of an organometallic derivative of type MR₁ (derived from R₁halogeno) (preferably Li or Mg) in an inert solvent under anhydrousconditions (e.g., THF) at low temperature (preferably -20° C.) tochlorodifluoroacetic acid (60a)

The aforementioned ketone (61) is added to a mixture of zinc, titaniumtetrachloride and desired aldehyde* at 0° C. under nitrogen in THF.

In step c, the alcohol functionality of the peptido alcohol of structure(62) is then oxidized by techniques and procedures well known andappreciated by one of ordinary skill in the art, such as a Swernoxidation using oxalyl chloride and dimethylsulfoxide, to give thecompounds of formula (63). Reference: D. Schirlin et al., Bioorg. andMedicinal Chem. Letters, 3 (1993), 253-258.

The following examples present typical syntheses. These examples areunderstood to be illustrative only and are not intended to limit thescope of the present invention in any way. As used herein, the followingterms have the indicated meanings: "g" refers to grams; "mmol" refers tomillimoles; "mL" refers to milliliters; "bp" refers to boiling point; "°C" refers to degrees Celsius; "mm Hg" refers to millimeters of mercury;"μL" refers to microliters; "μg" refers to micrograms; and "μM" refersto micromolar; "Z" or "Cbz" means carbobenzyloxy; "THF" meanstetrahydrofuran; "DCU" means N,N-dichlorourethane; "Eq" meansequivalent; "Atg" means gram atoms.

EXAMPLE 1 ##STR38## Preparation of Benzyloxycarbonyl-L-valyl-cyclopentylglycinal Step A: Benzyloxycarbonyl-cyclopentyl glycinol

A solution of borane-dimethylsulfide complex (1 M in dichloromethane,2.4 mL) is added dropwise under an atmosphere of nitrogen to a wellstirred solution of CbZ-cyclopentylglycine (0.336 g, 1.2 mmol) in 3 mLof anhydrous tetrahydrofuran. The resulting solution is stirred at roomtemperature for 16 hours. Water (1 mL) is carefully added and themixture evaporated. The oily residue is dissolved in ethyl acetate andthe solution washed with saturated solutions of citric acid, sodiumbicarbonate, and brine. The organic layer is dried over magnesiumsulphate, filtered, and evaporated to afford 0.2 g (60%) of the alcoholas an oil. R_(f) =0.25 (silica gel, ethyl acetate: petroleum ether 3:1)MS: MH⁺ =264.

Step B: Cyclopentyl glycinol

A mixture of the alcohol of Example 1, Step A (0.74 mmol) and 0.05 g ofpalladium hydroxide on carbon (Pearlman's catalyst, 10%) in 20 mL ofisopropanol is hydrogenated at room temperature and atmospheric pressurefor 16 hours. Filtration from the catalyst and evaporation of thefiltrate affords 0.076 g (80%) of the unprotected amino alcohol.

Step C: Benzyloxycarbonyl-L-valyl-cyclopentylglycinol

A solution of cyclopentylglycinol (0.065 g, 0.5 mmol) in 5 mLdichloromethane is added to a solution ofbenzyloxycarbonyl-L-valyl-0-benzotriazolyl ester [prepared by usualactivation of Cbz-valine (0.13 g, 0.5 mmol) with hydroxy-benzotriazole(0.077 g, 0.5 mmol), dicyclohexyl carbodiimide (0.103 g, 0.5 mmol) andN-methyl morpholine (0.101 g, 1 mmol)] in 5 mL of anhydrousdichloromethane. The resulting mixture is stirred for 16 hours at roomtemperature and filtered. The filtrate is evaporated and the oilyresidue dissolved in ethyl acetate. The solution is washed withsaturated solutions of citric acid, sodium bicarbonate, and brine.Drying over MgSO₄ and evaporation of solvents give 0.13 g of a paleyellow oil which is subjected to flash chromatography on silica gel(ethyl acetate: petroleum ether 1:1, R_(f) =0.2). Evaporation of thepooled product-containing fractions yields 0.13 g (70%) of the dipeptidealcohol as an oil. MS: MH⁺ =363.

Step D: Benzyloxycarbonyl-L-valyl-cyclopentyl glycinal

A mixture of the above dipeptide alcohol (0.086 g, 0.24 mmol),Dess-Martin periodinane (0.2 g, 0.48 mmol), and 4 mL of anhydrousdichloromethane is stirred at room temperature for 2 hours. Isopropanol(1 mL) is added and the solution evaporated to dryness. The solidresidue is applied to flash chromatography (silica gel, ethyl acetate:petroleum ether 1:7, R_(f) =0.3). Evaporation of the pooled,product-containing fractions affords 0.038 g (47%) of the title compoundas a solid.

Anal. Calcd for C₂₀ OH₂₈ O₄ N₂.025 H₂ O: C, 65.90; H, 7.74; N, 7.68.Found: C, 66.04; H, 7.62; N, 7.66.

EXAMPLE 2 ##STR39## Preparation of Benzyloxycarbonyl-L-valyl-cyclohexylglycinal Step A: 2-cyclohexylglycinol

A mixture of L-phenylglycine (0.96 g, 7 mmol), 0.1 g of 5% rhodium oncharcoal, and 50 mL acetic acid is hydrogenated at room temperatureunder a pressure of 8 bar for 48 hours. After filtration from thecatalyst the solution is evaporated to dryness (several evaporationswith carbon tetrachloride as cosolvent) to give an oil. Treatment ofthis oil with a saturated hydrochloric acid gas-ether solution andevaporation of solvent affords the title compound as a white solid(0.1.26 g, 100%).

Step B: Benzyloxycarbonyl-L-valyl-cyclohexyl glycinol

The title compound is obtained in 47% yield from the compound of Example2, Step A, and benzyloxycarbonyl-L-valine using the coupling proceduredescribed in Example 1, Step C. R_(f) =0.3 (silica gel, ethyl acetate:petroleum ether 4:6)

Anal. Calcd for C₂₁ H₃₂ O₄ N₂ : C, 66.99; H, 6.57; N, 7.44 Found: C,67.17; H, 6.76; N, 7.61.

Step C: Benzyloxycarbonyl-L-valyl-cyclohexyl glycinal

The title aldehyde is obtained in 78% yield from the alcohol of Example2, Step B, using the Dess-Martin oxidation procedure described inExample 1, Step D. R_(f) =0.5 (silica gel, ethyl acetate:petroleum ether2:3) MS: MH⁺ =375

Anal. Calcd for C₂₁ H₃₀ N₂ O₄ 0.0.25 H₂ O: C, 66.55; H, 6.11; N, 7.39Found; C, 66.35; H, 6.12; N, 7.56.

EXAMPLE 3 ##STR40## Preparation of CBz-L-Ala-L-Phe-H

Dissolve CBz-Ala-Phe-OH (0.5 g, 1.4 mmol) in methylene chloride (10 mL).Add N,N'-diisopropylethylamine (DIEA, 0.23 mL) and cool the solution to0° C. Add bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl, 0.34g), N,O-DimethylhydroxylamineeHCl (0.15 g, 1.5 mmol) and DIEA (0.46 mL)to the reaction. Stir for 2 hours and then pour into water. Extract theaqueous with ethyl acetate, dry the combined organic extracts overanhydrous sodium sulfate, filter and concentrate under vacuum. Purifythe residue by flash chromatography (30% ethyl acetate/hexane, silicagel) to provide the hydroxamate (0.23 g).

Dissolve the above prepared hydroxamate (16 g) in diethyl ether (10 mL)and cool the solution to 0° C. Add lithium aluminum hydride (0.20 g) andstir the reaction at -2° C. for 25 minutes. Then quench the reaction bydropwise addition of 10% aqueous potassium hydrogen sulfate and pourinto water (100 mL). Extract the aqueous with diethyl ether (3×50 mL).Combine the organic extracts, dry over anhydrous sodium sulfate, filterand concentrate under vacuum. Purify the residue by flash chromatography(30% ethyl acetate/hexane, silica gel) to provide the title compound(0.05 g).

EXAMPLE 4 ##STR41## Preparation of CBz-L-Phe-L-Phe-H

Dissolve CBz-Phe-Phe-OH (2.14 g, 4.8 mmol, obtained from BachemBioscience) and N-methylmorpholine (1.6 mL, 14.4 mmol) in methylenechloride (22 mL). Cool the solution to -22° C. and add isobutylchloroformate (0.62 mL, 4.8 mmol). Stir the reaction for 30 minutes andadd N,O-dimethylhydroxylamineeHCl (0.6 g, 6.2 mmol). Stir the reactionfor 45 minutes at -22° C. and then warm to room temperature and stirovernight. Pour the reaction into dilute aqueous hydrochloric acid andextract the aqueous with diethyl ether (2×200 mL). Combine the organicextracts and wash with saturated sodium bicarbonate, saturated sodiumchloride, dry over anhydrous sodium sulfate, filter and concentrateunder vacuum to provide the hydroxamate (1.81 g).

Dissolve the above prepared hydroxamate (1.0 g, 2.0 mmol) in THF (60 mL)and cool the solution to 0° C. Add lithium aluminum hydride (0.21 g, 5.5mmol) and stir the reaction for 40 minutes. Then quench the reaction bydropwise addition of 10% aqueous potassium hydrogen sulfate(approximately 3 mL). Pour the reaction mixture into water (300 mL).Extract the aqueous phase with diethyl ether (2×200 mL), dry thecombined organic extracts over anhydrous sodium sulfate, filter andconcentrate under vacuum. Crystallize the residue from ethylacetate/hexane to provide the title compound (0.25 g).

EXAMPLE 5 ##STR42## Preparation ofN-(N-Morpholylcarbonyl)-L-phenylalanyl-L-(O-benzyl) threoninal amide

Dissolve Boc-Thr(Bn)-OH (5.00 g, 16.3 mmol, obtained from BachemBioscience) in methylene chloride (65 mL). Then add consecutively1-hydroxybenzotriazole hydrate (2.20 g, 16.3 mmol),N,O-dimethylhydroxylamineHCl (1.59 g, 16.3 mmol), N-methylmorpholine(1.79 mL, 16.3 mmol) and 1-(3-dimethylmorpholinel)3-ethylcarbodiimideHCl (3.10 g, 16.3 mmol). Stir the reaction for 1hour under an atmosphere of nitrogen. Then dilute the reaction with 10%aqueous hydrochloric acid (200 mL) and extract with methylene chloride(135 mL). Wash the separated organic layer with 10% aqueous hydrochloricacid (100 mL), saturated sodium bicarbonate (100 mL), saturated sodiumchloride (100 mL), dry over anhydrous magnesium sulfate, filter andconcentrate under vacuum to provide the desired hydroxamate (4.83 g) asa clear colorless oil.

Dissolve the above prepared hydroxamate (4.80 g, 13.6 mmol) in ethylacetate (270 mL) and cool the solution to 0° C. under an atmosphere ofnitrogen. Bubble hydrogen chloride (gas) through the solution for 50minutes. Then bubble nitrogen through the solution as it warms to roomtemperature. Concentrate under vacuum, add hexane (100 mL) and againconcentrate under vacuum to provide the HCl salt of the amide (3.64 g)as a white foam.

Dissolve Boc-Phe-OH (3.22 g, 12.12 mmol) in methylene chloride (48 mL).Add consecutively 1-hydroxybenzotriazole hydrate (1.64 g, 12.12 mmol),the above prepared HCl salt of the hydroxamate (3.50 g, 12.12 mmol),N-methylmorpholine (1.23 mL, 12.12 mmol) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimideHCl (2.23 g, 12.12 mmol)and stir the reaction overnight at room temperature under an atmosphereof nitrogen. Then dilute the reaction with methylene chloride (100 mL)and add 10% aqueous hydrochloric acid (150 mL). Separate the layers andwash the organic layer with 10% aqueous hydrochloric acid (2×75 mL),saturated sodium bicarbonate (2×75 mL), saturated sodium chloride (75mL), dry over anhydrous magnesium sulfate, filter and concentrate undervacuum to provide the coupled amide (5.19 g) as a white foam.

Dissolve the above prepared coupled amide (5.00 g, 10.01 mmol) in ethylacetate (200 mL) and cool the solution to 0° C. under an atmosphere ofnitrogen. Bubble hydrogen chloride (gas) through the solution for 1hour. Then bubble nitrogen through the solution as it warms to roomtemperature. Concentrate under vacuum, add hexane (100 mL) and againconcentrate under vacuum. Dry the residue over potassium hydroxide toprovide the HCl salt of the coupled amide (quantitative yield).

Dissolve the above prepared HCl salt of the coupled amide (0.750 g, 1.72mmol) in methylene chloride (34 mL). Add morpholine chloride (0.399 mL,3.44 mmol) and N-methylmorpholine (0.389 g, 3.44 mmol). Stir thereaction at room temperature under an atmosphere of nitrogen forapproximately 2 hours. Concentrate the reaction under vacuum and purifythe residue by flash chromatography (80% ethyl acetate/acetone, silicagel) to provide the morpholine carboxamide (0.430 g) as a white foam.

Dissolve the above prepared morpholine carboxamide (0.4 g, 0.780 mmol)in THF (7.8 mL) and cool to 0° C. under an atmosphere of nitrogen. Addlithium aluminum hydride (36.9 g, 0.975 mmol) and stir the reaction for1 hour at 0° C. Quench the reaction by addition of 10% potassiumhydrogen sulfate add ethyl acetate (20 mL) and 10% aqueous hydrochloricacid (20 mL). Separate the layers and wash the organic layer with 10%aqueous hydrochloric acid (2×15 mL), saturated sodium bicarbonate (15mL), saturated sodium chloride (15 mL), dried over anhydrous magnesiumsulfate, filtered and concentrated under vacuum to provide the titlecompound (0.264 g) as a white foam.

EXAMPLE 6 ##STR43## Preparation of2-(N-Benzyloxycarbonyl-L-valyl)amino-4,4-difluoro-1,7-diphenyl-3,5-dioxoheptane

Step A : N-Benzyloxycarbonyl-L-valyl-L-phenylalanine, N,O-dimethylhydroxamate

Add N,N'-dicyclohexylcarbodiimide (0.920 g, 4.5 mmol) to a solution ofCBz(L)-Val-Phe-OH (0.220 g, 0.55 mmol) and hydroxybenzotriazole (0.93 g,0.61 mmol) in methylene chloride (12 mL). Stir at 0° C. for 1 hour. AddN,O-dimethyl-hydroxylamineHCl (0.59 g, 0.61 mmol) andN-methylmorpholine (0.61 g, 0.61 mmol) to the reaction and stir at 25°C. for 12 hours. Filter the mixture, wash with methylene chloride andconcentrate the filtrate under vacuum to provide the crude amide as anoil. Purify the crude residue by flash chromatography (silica gel, 2:8ethyl acetate:cyclohexane) to provide the title compound (0.250 g).

Step B: N-Benzyloxycarbonyl-L-valyl-L-phenylalaninal

Add the above prepared hydroxamate (0.220 g, 5 mmol) to a solution oflithium aluminum hydride (0.19 g, 0.49 mmol) in diethyl ether (10 mL) at0° C. under inert atmosphere. Stir for 30 minutes, allow the reaction towarm to room temperature and stir for 1 hour. Separate the phases andwash the organic phase with saturated sodium carbonate (10 mL),saturated sodium chloride, dry over magnesium sulfate, filter andconcentrate under vacuum to provide the title compound CBz(L)-Val-Phe-H(0.179 g) used without further purification.

Step C:2-(N-Benzyloxycarbonylvalyl)amino-4,4-difluoro-1,7-diphenyl-3-hydroxy-5-oxoheptane

Add titanium tetrachloride (0.019 g, 0.1 eq) to a suspension ofactivated zinc (0.196 g, 3mAtg) in anhydrous THF (3 mL) at 0° C. undernitrogen. Stir 30 minutes and add CBz(L)-Val-Phe-H (0.42 g, 1.1 mmol),1-chloro-1,1-difluoro-2-oxo-4-phenylbutane (0.218 g, 1.1 mmol), inanhydrous THF (4 mL). Allow the reaction to warm to room temperature andstir for 12 hours. Add a saturated solution of ammonium chloride (2 mL).Extract two times with diethyl ether (4 mL) and wash the organic phasewith brine and dry over anhydrous magnesium sulphate. Concentrate theorganic phase under vacuum. Purify the crude residue by flashchromatography (silica gel, 3:7 ethyl acetate:cyclohexane) to providethe title alcohol (0.381 g).

Step D:2-(N-Benzyloxycarbonyl-L-valyl)amino-4,4-difluoro-1,7-diphenyl-3,5-dioxoheptane

Add dimethylsulfoxide (0.331 g, 4.26 mmol) to a solution of oxalylchloride (0.269 g, 2.13 mmol) in anhydrous methylene chloride (2 mL) at-55° C. under nitrogen. Stir for ten minutes at -55° C. and add theabove prepared alcohol (0.3 g, 0.53 mmol) in anhydrous methylenechloride (2 mL). Stir the reaction 2 hours at this temperature and allowthe reaction to warm to -20° C. Add triethylamine (0.321 g, 1.68 mmol)and allow the reaction to warm to room temperature. Stir the mixture anadditional few minutes. Dilute with ethyl acetate (10 mL). Wash theorganic phase with hydrochloric acid (3×3 mL, 0.1N) and saturatedaqueous ammonium chloride. Dry the organic phase over anhydrousmagnesium sulfate, filter and concentrate under vacuum to provide thecrude compound (0.22 g).

Purification of the crude material by crystallization provides the titlecompound (0.102 g).

Anal. Calcd: 67.95; H, 6.24; N, 4.95. Found: C, 66.99; H, 6.15; N, 5.30.

EXAMPLE 7 ##STR44## Preparation of4-(N-Phenylpropionyl-L-valyl)amino-2,2-difluoro-3-oxo-5-phenyl-N-benzylpentanamide

Step A: N-Benzyloxylcarbonyl-phenylalaninal

The aldehyde CBz(L)-Phe-H (4.19 g) is obtained by reduction ofCBz(L)-Phe-OH (15.00 g, 50 mmol) following the procedure described inExample 6 (step A and B).

Step B: 4-Benzyloxycarbonylamino-2,2-difluoro-3-hydroxy-5-phenylpentanoic acid, ethyl ester

Add CBz-(L)-Phe-H (4.19 g, 14.8 mmol) and ethylbromo-difluoroacetate(6.30 g, 31 mmol) in dry THF (40 mL) to a refluxing suspension ofactivated zinc wool (2.0 g, 31 mAtg) in dry THF (10 mL) to maintain agentle reflux of the mixture. Stir the solution 12 hours at roomtemperature. Add to the mixture ethyl acetate (100 mL), brine (20 mL),potassium hydrogenosulfate (20 mL). Extract the aqueous phase with ethylacetate (3×60 mL), dry over magnesium sulfate and concentrate undervacuum. Purify the crude residue by flash chromatography (silica gel,3:7 ethyl acetate:cyclohexane) to provide the title compound (3.70 g).

Step C:4-Benzyloxycarbonylamino-2,2-difluoro-3-hydroxy-5-phenyl-N-benzylpentanamide

Add a solution of benzylamine (1.93 g, 18 mmol) in THF (10 mL) to asolution of the ethyl ester (1.42 g, 3.5 mmol) in THF (10 mL). Stir 12hours. Add ethyl acetate (100 mL), wash with a 0.1 N aqueoushydrochloric acid (2×100 mL) and with water (100 mL) and brine (100 mL).Dry over anhydrous magnesium sulfate. Purification of the crude materialby flash chromatography (silica gel, 2:8 ethyl acetate:cyclohexane)provides the title compound (1.16 g).

Step D: 4-Amino-2,2-difluoro-3-hydroxy-5-phenyl-N-benzyl pentanamide

Add the above prepared amide (0.81 g, 1.70 mmol) to a suspension of 10%palladium on carbon (0.28 g) in absolute ethanol (75 mL). Stir for 12hours, under atmospheric pressure of hydrogen. Filtrate the catalyst,wash with ethanol and concentrate under vacuum to provide the titledeprotected amine (0.5 g).

Step E:4-(N-Phenylpropionyl-L-valyl)amino-2,2-difluoro-3-hydroxy-5-phenyl-N-benzylpentanamide

Add N,N'-dicyclohexylcarbodiimide (0.165 g, 0.80 mmol) to a solution ofhydrocinnamoyl-Val-OH (3-phenylpropionyl-Val-OH) (0.199 g, 0.80 mmol) inanhydrous acetonitrile (15 mL). Stir at 0° C. for 1 hour. Add the aboveprepared amine (0.210 g, 0.80 mmol) to the reaction and stir at 25° C.for 12 hours. Filter the mixture, wash with ethyl acetate andconcentrate the filtrate under vacuum to provide the crude amide as anoil. Purify the crude residue by flash chromatography (silica gel, 2:8ethyl acetate:cyclohexane) to provide the title compound (0.16 g).

Step F:4-(N-Phenylpropionyl-L-valyl)amino-2,2-difluoro-3-oxo-5-phenyl-N-benzylpentanamide

Add the above prepared alcohol (0.145 g, 0.25 mmol) in methylenechloride (10 mL) and tert-butyl alcohol (0.055 g, 0.75 mmol) to asuspension of Dess-Martin reagent (0.318 g, 0.75 mmol) in methylenechloride. Stir 12 hours at room temperature and concentrate the mixtureunder vacuum. Purify the crude residue by flash chromatography (silicagel, 3:7 ethyl acetate:cyclohexane) to provide the title compound (0.063g).

Anal. Calcd for C₃₂ H₃₅ N₃ O₄ F₂ : C, 68.19; H, 6.26; N, 7.45 Found: C,67.90; H, 6.30; N, 7.33.

EXAMPLE 8 ##STR45## Preparation of4-(N-Benzyloxycarbonyl-L-valyl)amino-2,2-difluoro-3-oxo-4-phenyl-N-(trimethylsilylmethyl)pentanamide

Prepare the4-benzyloxycarbonylamino-2,2-difluoro-3-hydroxy-5-phenylpentanoic acid,ethyl ester as described in Example 7, Step B.

Step A:4-Benzyloxycarbonylamino-2,2-difluoro-3-hydroxy-5-phenyl-N-trimethylsilylmethylpentanamide

Add the above prepared difluoro alcohol (0.25 g, 0.61 mmol) totrimethylsilylmethylamine (0.81 mL, 6.10 mmol) in THF (2.5 mL). Stir andheat under reflux for 12 hours. Concentrate under vacuum. Dilute with anaqueous solution of potassium hydrogenosulphate (5 mL) and extract withdiethyl ether (3×5 mL). Wash the organic phase with water (2×15 mL). Dryover sodium sulphate. Purify the crude residue by flash chromatography(silica gel, 25:75 ethyl acetate:cyclohexane) to provide the titlecompound (0.212 g).

Step B;4-Amino-2,2-difluoro-3-hydroxy-5-phenyl-N-(trimethylsilylmethyl)pentanamid

Add the above prepared amide (0.10 g, 0.22 mmol) to a suspension of 10%palladium on carbon (0.10 g) in absolute ethanol (10 mL). Stir for 12hours under atmospheric pressure of hydrogen. Filter the catalyst, washwith ethanol and concentrate under vacuum to provide the titledeprotected amine (0.71 g).

Step C:4-(N-Benzyloxycarbonyl-L-valyl)amino-2,2-difluoro-3-hydroxy-5-phenyl-N-(trimethylsilylmethyl)pentanamide

Add N,N'-dicyclohexylcarbodiimide (0.045 g, 0.22 mmol) to a solution ofCBz(L)-Val-OH (0.055 g, 0.22 mmol) and hydroxybenzotriazole (0.03 g,0.22 mmol) in dimethyl-formamide (10 mL). Stir at 0° C. for 30 minutes.Add the above prepared amine (0.073 g, 0.22 mmol) to the reaction andstir at 25° C. for 12 hours. Add water and brine. Extract with ethylacetate, wash with water, dry over sodium sulphate and concentrate.Dilute with acetonitrile (5 mL) and precipitate DCU. Filter andconcentrate the filtrate under vacuum to provide the crude amide. Purifythe crude residue by flash chromatography (silica gel, 35:65 ethylacetate:petroleum ether) to provide the title compound (0.40 g).

Step D:4-(N-Benzyloxycarbonyl-L-valyl)amino-2,2-difluoro-3-oxo-5-phenyl-N-(trimethylsilylmethyl)pentanamide

Add dimethylsulfoxide (0.031 g, 0.44 mmol) in anhydrous methylenechloride (1 mL) to a solution of oxalyl chloride (0.02 mL, 0.22 mmol) inmethylene chloride (1 mL) at -60° C. Stir for 5 minutes at -60° C. andadd the above prepared alcohol (0.041 g, 0.07 mmol) in methylenechloride (2 mL). Stir the reaction 1 hour at this temperature. Add thetriethylamine (0.09 mL, 0.65 mmol) and allow the reaction to warm toroom temperature. Stir the mixture an additional few minutes. Dilutewith methylene chloride (10 mL). Wash the organic phase with potassiumhydrogenosulphate (3×10 mL, 1N) and water (2×10 mL). Dry the organicphase over sodium sulfate, filter and concentrate under vacuum toprovide the crude compound. Purify the crude residue by flashchromatography (ethylacetate:petroleum ether 25:75, R_(f) : 0.2) toprovide the title compound (0.015 g).

Anal. Calcd for C₃₁ H₃₃ N₃ O₅ F₂, 0.25 H₂ O: C, 58.93; H, 6.71; N, 7.36Found: C, 58.74; H, 6.72; N, 7.16

EXAMPLE 9 ##STR46## Preparation of3-(benzyloxYcarbonyl-L-valyl)amino-1,1,1,-trifluoro-2-oxo-4-phenylbutane

Prepare the3-[benzyloxycarbonyl-L-valyl]amino-1,1,1,-trifluoro-2-hydroxy-4-phenylbutanefrom CBz(L)-Val-OH (0.128 g, 0.5 mmol) and1,1,1-trifluoro-3-amino-4-phenyl-2-butanol (0.112 g, 0.5 mmol, J. Med.Chem. 1990, 33, 394-407) by the coupling reaction described in Example6.

Oxidize the above prepared alcohol (0.71 g, 0.16 mmol) using oxalylchloride as described in Example 8 to provide the title compound (0.60g).

Anal. Calcd for C₂₃ H₂₅ F₂ N₂ O₄ F₂, 0.5 H₂ O: C, 60.12; H, 5.70; N,6.10 Found: C, 60.08; H, 5.81; N, 5.89.

EXAMPLE 10 ##STR47## Preparation of1-N-Benzyloxycarbonylvalylamino-2-phenyl-ethane boronic acid

Desilylate(+)pinanediol-1-N,N-bis(trimethylsilyl)amino-2-phenylethaneboronate (J.Am. Chem. Soc. (1981) 103,5241) with methanol at 0° C. Concentrate undervacuum and wash with diethyl ether to provide the crude deprotectedaminoboronate.

Add N-carbobenzoxyvaline anhydride to the solution of the crudedeprotected aminoboronate prepared above in anhydrous THF to yield thecoupled compound.

Cleave the pinanediol of the coupled compound prepared above using borontrichloride as described in the procedure J. Am. Chem. Soc. (1980), 102,7590.

Purify the title compound by ion exchange column or by the proceduredescribed in Biochemistry (1987), 26, 7609 and references herein.

EXAMPLE 11 ##STR48## Peparation ofN-[2-[4-(Aminoiminomethyl)amino)phenyl]-1-trifluoroacetvlethyl]benzamide,hydrochloride hydrate

The synthesis for this compound is described in Liebigs Ann. Chem. 1990,1-6 which is incorporated herein by reference.

EXAMPLE 12 ##STR49## Preparation of2-(N-Benzyloxycarbonyl-L-valyl)amino-3-trimethylsilylpropanal

Step A: 2-Benzyloxycarbonylamino-3-trimethylsilyl-propanoic acid, methylester

A solution of 5.58 g (25 mmol) of N-benzyloxycarbonyl glycine methylester in dry THF (70 ml) is added dropwise to a solution at -78° C. oflithium diisopropylamine (8.76 ml, 62.5 mmol) and tetramethylethylenediamine (9.43 ml, 62.5 mmol) in dry THF (100 ml), under nitrogen. Afterthe addition is complete, the solution is stirred for 2 hours at -78°C., then 15 minutes at -30° C. and cooled to -78° C. A solution of 5.36g (25 mmol) of iodomethyl-trimethylsilane in dry hexamethylphosphoramide(39 ml) is added dropwise to the resultant syrupy mixture. After theaddition is complete, the reaction mixture is warmed up to -50° C., keptat this temperature for 1 hour and cooled to -78° C., just beforehydrolysis. The reaction mixture is quenched by addition of water andammonium chloride and diluted with ether. The organic layer is washedwith 1N potassium hydrogen sulfate, twice with water and dried oversodium sulfate. The solvent is evaporated and the residue obtained (8.03g) is purified by flash chromatography (silica gel,ethylacetate/petroleum ether: 2/8). 4.30 g of the title compound are obtained(yield: 56%) (colorless oil). R_(f) : 0.51 (ethyl acetate/petroleumether: 2/8).

Step B: 2-Amino-3-trimethylsilylpropanoic acid, methyl ester,hydrochloride

A solution of the derivative of Example 12, Step A (0.309 g, 1 mmol) inethanol (30 ml) and in dry diethylether saturated in hydrogen chloride(1.5 ml) is stirred at room temperature for 24 hours under an atmosphereof hydrogen in the presence of 10% Palladium on charcoal (0.03 g). Thehydrogen atmosphere is changed to a nitrogen atmosphere and the catalystfiltered off. After concentration under vacuo, the title compoundobtained as a solid is used as such in the next step.

Step C: 2-(N-Benzyloxycarbonyl-L-valyl)amino-3-trimethyl-silylpropanoicacid, methyl ester

To a stirred solution of N-benzyloxy-carbonyl-L-valine (0.311 g, 1.24mmol), 1-hydroxybenzotriazole, hydrate (0.167 g, 1.24 mmol) andN,N'-dicyclohexylcarbodiimide (0.255 g, 1.24 mmol) in methylene chloride(20 ml) and dimethylformamide (3 ml), at 0° C. are successively addedthe amine of Example 12, Step B (0.262 g, 1.24 mmol) andN-methylmorpholine (0.136 ml, 1.24 mmol). The cooling bath is removedand the reaction mixture is stirred at room temperature for 17 hours.The reaction mixture is then filtered off and the filtrate wisconcentrated under vacuo. The residue (0.476 g) is purified by flashchromatography (silica gel, petroleum ether/ethyl acetate: 8/2; R_(f) :0.14) to give the title compound (0.316 g, 77% yield for two steps).

Step D: 2-(N-Benzyloxycarbonyl-L-valyl)amino-3-trimethyl-silylpropanal

A solution of 1M diisobutylaluminum hydride in hexane (1.55 ml) is addeddropwise to a solution of the ester of Example 12, Step C, at -78° C.(0.316 g, 0.77 mmol) in anhydrous ether (7.5 ml) and anhydrous toluene(3.5 ml), under nitrogen. The solution is stirred at -78° C. for 45minutes and hydrolyzed slowly with a saturated solution of ammoniumchloride in water. The aqueous phase is extracted twice with ether (2×20ml) and the organic phases washed successively with iN potassiumhydrogen sulfate (10 ml) and water (20 ml). The combined organic layersare dried over sodium sulfate, filtered off and removal of the solventunder vacuo affords a solid residue (0.260 g) which is purified by flashchromatography (silica gel, petroleum ether/ethyl acetate: 75/25, R_(f): 0.25) to give the title compound in 53% yield (0.15 g).Crystallization from dichloromethane/pentane gives 0.077 g of a whitecottony solid.

Analysis calcd for C₁₉ H₃₀ N₂ O₄ Si: C, 60.29; H, 7.99; N, 7.40. Found:C, 60.23; H, 8.10; N, 7.42.

EXAMPLE 13 ##STR50## Preparation of2-(N-Benzyloxycarbonyl-L-valyl)amino-3-phenyldimethylsilyl-propanal

Step A: 2-tert-Butoxycarbonylamino-3-phenyldimethylsilyl-propanoic acid,methyl ester

The title ester is prepared in 28% yield from N-tert-butoxycarbonylglycine methyl ester and iodomethyl-phenyldimethylsilane (prepared in81% yield from commercially available chloromethyl-phenyldimethylsilane)following the procedure described in Example 12, Step A. R_(f) : 0.23(silica gel, ethyl acetate/petroleum ether: 1/9).

Step B: 2-Amino-3-phenyldimethylsilane-propanoic acid, methyl ester

A solution of the derivative of Example 13, Step A (0.92 g, 2.73 mmol)in formic acid (30 ml) is kept for 2 hours at room temperature. Afterremoval of formic acid in vacuo, the residue is dissolved in ethylacetate (20 ml), extracted with 1M sodium carbonate (20 ml) and washedtwice with water, the aqueous phases being extracted once more withethyl acetate (20 ml). After drying of the combined organic layers oversodium sulfate, the solvent is evaporated and the title compound isobtained in 98% yield (0.64 g).

Step C:2-(N-Benzyloxycarbonyl-L-valyl)amino-3-phenyl-dimethylsilyl-propanoicacid, methyl ester

The title compound is obtained from the amine of Example 13, Step B andN-benzyloxycarbonyl-L-valine using the coupling method given in Example12, Step C but with 1-ethyl -3(3-dimethylaminopropyl)carbodiimide,hydrochloride instead of N,N'-dicyclohexylcarbodiimide (74% yield).R_(f) : 0.19 (silica gel, ethyl acetate/petroleum ether: 2/8).

Step D:2-(N-Benzyloxycarbonyl-L-valyl)amino-3-phenyldimethylsilyl-propanal

The title aldehyde is prepared in 33% yield from the ester of Example13, Step C following the reduction procedure described in Example 12,Step D. R_(f) : 0.17 (silica gel, ethyl acetate/petroleum ether: 2/8).

Anal. Calcd for C₂₄ H₃₂ N₂ O₄ Si: C, 65.42; H, 7.32; N, 6.36 Found: C,65.33; H, 7.10; N, 6.26.

EXAMPLE 14 ##STR51## Preparation of2-(N-Benzyloxycarbonyl-L-valyl)amino-3-vinyldimethylsilyl-propanal

Step A; 2-tert-Butoxycarbonylamino-3-vinyldimethylsilyl-propanoic acid,methyl ester

The title ester is prepared in 51% yield from N-tert-butoxycarbonylglycine, methyl ester and iodomethyl-vinyldimethylsilane following theprocedure given in Example 12, Step A.

R_(f) : 0.35 (silica gel, ethyl acetate/petroleum ether: 1/9).

Step B: 2-Amino-3-vinyldimethylsilyl-propanoic acid, methyl ester

The title amine is obtained from the derivative of Example 14, Step Afollowing the deprotection method given in Example 13, Step B(quantitative yield).

Step C: 2-(N-Benzyloxycarbonyl-L-valyl)amino-3-vinyldimethylsilylpropanoic acid, methyl ester

The title compound is prepared in 68% yield from the amine of Example14, Step B and N-benzyloxycarbonyl-L-valine using the coupling methoddescribed in Example 13, Step C.

R_(f) =0.22 (silica gel, ethyl acetate: petroleum ether 2:8) MS: MH⁺=421, MNH₄ ⁺ =438

Step D:3-(N-Benzyloxycarbonyl-L-valyl)amino-3-vinyl-dimethylsilyl-propanal

The title aldehyde is obtained from the ester of Example 14, Step Cfollowing the reduction procedure given in Example 12, Step D.

EXAMPLE 15 ##STR52## Preparation ofN-Benzyloxycarbonyl-L-valyl-(O-methyl)-L-tyrosinal Step A:N-Benzyloxycarbonyl-L-valyl-O-methyl-L-tyrosine benzyl ester

To a solution of N-benzyloxycarbonyl-L-valine anhydride (0.339 g, 0.7mmol) in anhydrous dichloromethane (15 ml) are addedO-methyl-L-tyrosine, benzyl ester, toluene-4-sulfonate (0.330 g, 0.7mmol) and N-methyl morpholine (0.081 g, 0.8 mmol). The reaction isstirred at room temperature overnight. The solvent is removed invacuoand the residue is purified by flash chromatography (silica gel: 2:8ethyl acetate/cyclohexane) to give the title compound as a white solid.

Step B: N-Benzyloxycarbonyl-L-valyl-(O-methyl)-L-tyrosinal

To a solution of N-benzyloxycarbonyl-L-valyl-o-methyl-L-tyrosine benzylester (0.250 g, 0.48 mmol) in anhydrous toluene (5 ml) and diethyl ether(5 ml) at -78° C., is added a 1.2 M solution of diisobutyl aluminumhydride in hexane. (1.6 ml, 2 mmol). The reaction is stirred at -78° C.for one hour then hydrolized with a saturated solution of potassiumsodium tartrate (5 ml). The temperature is then allowed to rise to roomtemperature.

The mixture is acidified with a 1M solution of potassiumhydrogenosulfate until pH˜3 and extracted three times with ethyl acetate(3×20 ml). The organic layer is dried over anhydrous magnesium sulfate.Filtration and removal of the solvent invacuo affords a residue which ispurified by flash chromatography (silica gel: 3:7 ethylacetate/cyclohexane) to give the title compound as a white solid.

EXAMPLE 16 ##STR53## Preparation ofN-Benzyloxycarbonyl-L-valvl-O-benzyl-L-tyrosinal Step A:N-tert-Butoxycarbonyl-O-Benzyl-L-tyrosine,N,O-dimethyl hydroxamate

To a solution of tert-butoxycarbonyl amino-O-(L)benzyl tyrosine (23 g,61.9 mmol), in anhydrous methylene chloride (250 ml) at 0° C. are addedN,N-dicyclohexylcarbodiimide (12.75 g, 61.9 mmol) andhydroxybenzotriazole (9.47 g, 61.9 mmol). The mixture is stirred at 0°C. for 10 minutes, and N,O-dimethylhydroxylamine, HCl (6.04 g, 61.9mmol) and N-methylmorpholine (6.25 g, 61.9 mmol) are then added. Thereaction is stirred at room temperature for 12 hours. The mixture isthen filtered and the filtrate concentrated. The crude mixture ispurified by flash chromatography (silica gel: 2:8 ethylacetate:cyclohexane) to provide the title compound as a white solid(22.60 g, 88% yield). R_(f) ≅0.36 (ethyl acetate/cyclohexane).

Step B: O-Benzyl-L-tyrosine,N,O-dimethyl hydroxamate

A solution of N-tert-butoxycarbonyl-O-benzyl (L) tyrosine, N,O-dimethylhydroxamate (8.28 g, 20 mmol) in trifluoroacetic acid (100 ml) isstirred at 0° C. for 1 hour. The solvent is removed in vacuo. Theresidue is taken off in diethyl ether (250 ml) and washed three timeswith a saturated solution of sodium carbonate (3×50 ml). The organiclayer is dried over anhydrous magnesium sulphate. Filtration and removalof the solvent in vacuo yields the title compound as a pale yellow oil(6.00 g, 90% yield) which is used without purification in the next step.

Step C: N-Benzyloxycarbonyl-L-valyl-O-benzyl-L-tyrosine,N,O-dimethylhydroxamate

To a solution of Z-L valine anhydride (8.47 g, 17.5 mmol) in anhydrousmethylene chloride (150 ml) is addedO-benzyl-L-tyrosine,N,O-dimethylhydroxamate (5.50 g, 17.5 mmol). Themixture is stirred at room temperature overnight. The solvent is removedin vacuo and the crude mixture is purified by flash chromatography(silica gel: 2:7 ethyl acetate/cyclohexane) to provide the titlecompound as a white solid (8.90 g, 93% yield). R_(f) ≅0.18 (1:1ethylacetate/cyclohexane).

Step D: N-Benzyloxycarbonyl-L-valyl-O-benzyl-L-tyrosinal

To a solution of N-benzyloxycarbonyl-L-valyl-O-benzyl-L-tyrosine,N,O-dimethylhydroxamate (8.90 g, 16.2 mmol) in anhydrous diethyl ether(150 ml) and anhydrous THF (20 ml) at 0° C. is added lithium aluminumhydride (0.67 g, 17.7 mmol). The mixture is stirred at 0° C. for 1 hour.A solution of 1M potassium hydrogenosulphate (25 ml) is then added withprecaution. The organic layer is separated and the aqueous phase isextracted twice with ethyl acetate (2×100 ml). The combined organiclayers are then washed with a 3N solution of hydrochloric acid (30 ml),water (30 ml) and brine (30 ml), and dried over anhydrous magnesiumsulphate. Filtration and evaporation of the solvent in vacuo affords awhite solid which is recrystallized in ethylacetate/pentane to give thetitle compound (5.40 g, 68% yield).

R_(f) ≅0.33 (ethyl acetate/cyclohexane) m.p.: 160-162° C.

Analysis calculated for C₂₉ H₃₂ N₂ O₅ : Calc. C, 71.29; H, 6.60; N,5.73. Found: C, 71.18; H, 6.94; N, 6.30.

EXAMPLE 17 ##STR54## Preparation ofN-Benzyloxycarbonyl-2-cyclopentylglycine-O-benzyl-L-tyrosinal

Step A:N-Benzyloxycarbonyl-2-cyclopentylglycine-O-benzyl-L-tyrosine,N,O-dimethylhydroxamate

To a solution of racemic N-benzyloxycarbonyl-2-cyclo-pentylglycine(0.831 g, 3 mmol) in anhydrous acetonitrile (20 ml) is addedN-methylmorpholine (0.323 g, 3.2 mmol). The mixture is cooled to -20° C.under nitrogen and isobutyl-chlorformate 0.410 g, 3 mmol) is added.After 10 minutes stirring at -20° C., O-benzyl-L-tyrosine,N,O-dimethylhydroxamate (1.00 g, 3.1 mmol) in anhydrous acetonitrile (10 ml) isadded. The mixture is stirred at -20° C. under nitrogen for 4 hours andthen the temperature is allowed to rise to room temperature overnight.The crude mixture is evaporated and the residue is purified by flashchromatography (silica gel: 3:7 ethyl acetate/cyclohexane) to providethe title compound as a white solid (1.60 g, 93% yield). R_(f) ≅0.26(ethyl acetate/cyclohexane 1:1).

Step B: N-Benzyloxycarbonyl-2-cyclopentylglycine-O-benzyl-L-tyrosinal

To a solution ofN-benzyloxycarbonyl-2-cyclopentyl-glycine-O-benzyl-L-tyrosine,N,O-dimethylhydroxamate (1.60 g, 2.8 mmol) in anhydrous diethyl ether (20 ml) andanhydrous THF (20 ml) at 0° C. is added lithium aluminum hydride (0.121g, 3.2 mmol). The mixture is stirred at 0° C. for 1 hour. A solution of1M aqueous potassium hydrogeno-sulphate sulphate (25 ml) is added. Themixture is extracted three times with ethyl acetate (3×50 ml). Thecombined organic layer is washed with 3N hydrochloric acid, water andbrine, then dried over anhydrous magnesium sulphate. Filtration andevaporation of the solvent invacuo afforded a white solid which ispurified by recrystallization (Ethyl acetate/pentane) to give the titlecompound (0.96 g, 67% yield).

R_(f) ≅0.34 (ethyl acetate/cyclohexane 1:1) MS: [MH]⁺ =515 [MNH₄ ]³⁰=532

Analysis calculated for C₃₁ H₃₄ N₂ O₅ : Calc.: C, 72.35; H, 6.66; N,5.44. Found: C, 72.38; H, 6.64; N, 5.52.

EXAMPLE 18 ##STR55## Preparation ofN-Benzyloxycarbonyl-2-cyclohexylglycine-O-benzyl-L-tyrosinal

Step A:N-Benzyloxycarbonyl-2-cyclohexylglycine-O-benzyl-L-tyrosine,N,O-dimethylhydroxamate

To a solution of racemic N-benzyloxycarbonyl-2-cyclo-hexylglycine (0.873g, 3 mmol) in anhydrous acetonitrile (25 ml) is added N-methylmorpholine(0.323 g, 3.2 mmol). The mixture is cooled to -20° C. andisobutylchloroformate (0.410 g, 3 mmol) is added. After 10 minutesstirring at -20° C. under nitrogen, O-benzyl-L-tyrosine,N,O-dimethylhydroxamate (1.00 g, 3.1 mmol) in anhydrous acetonitrile (10 ml) isadded. The mixture is stirred at -20° C. under nitrogen for 4 hours andthen the temperature is allowed to rise to room temperature overnight.The solvent is removed inuacuo and the residue is purified by flashchromatography (silica gel: 3:7 ethyl acetate/cyclohexane) to providethe title compound as a white solid (1.00 g, 57% yield). R_(f) ≅0.35(ethyl acetate/cyclohexane).

Step B: N-Benzyloxycarbonyl-2-cyclohexylglycine-O-benzyl-L-tyrosinal

To a solution ofN-benzyloxycarbonyl-2-cyclohexyl-glycine-O-benzyl-L-tyrosine,N,O-dimethylhydroxamate (0.96 g, 1.6 mmol) in anhydrous diethyl ether (20 ml) andanhydrous THF (20 ml) at 0° C. is added lithium aluminum hydride (0.068g, 1.8 mmol). The mixture is stirred at 0° C. for 1 hour. An aqueoussolution of 1M aqueous potassium hydrogenosulphate (25 ml) is added. Themixture is extracted three times with ethyl acetate (3×50 ml). Thecombined organic layer is washed with 3N hydrochloric acid, water andbrine, then dried over anhydrous magnesium sulphate.

Filtration and evaporation of the solvent invacuo affords a white solidwhich is purified by recrystallization (AcOEt/pentane) to give the titlecompound (0.56 g, 67% yield).

MS: [EMH]⁺ =529 [MNH₄ ]⁺ =546 Analysis calculated for C₃₂ H₃₆ N₂ O₅ :Calc: C, 72.71; H, 6.86; N, 5.30. Found: C, 72.43; H, 6.92; N, 5.43.

EXAMPLE 19 ##STR56## Preparation ofN-Benzyloxycarbonyl-L-valyl-3-(1-naphthyl)-L-alaninal Step A:N-tert-Butoxycarbonyl-[3-(1-naphthyl)-L-alanine]-N,O-dimethylhydroxamate

To a solution of N-tert-butoxycarbonyl-[3-(1-naphthyl)-L-alanine] (0.65g, 2 mmol) in anhydrous dichloromethane (20 ml) at 0° C. are addedN,N-dicyclohexyl carbodiimide (0.412 g, 2 mmol) and hydroxybenzotriazole(0.306 g, 2 mmol). After 10 minutes stirring,N,O-dimethylhydroxyl-amine, chlorohydrate (0.195 g, 2 mmol) andN-methyl-morpholine (0.202 g, 2 mmol) are added. The reaction is stirredat room temperature for 12 hours. The mixture is filtered and thefiltrate concentrated. The crude residue is purified by flashchromatography (silica gel: 4:6 ethyl acetate/cyclohexane). The titlecompound is obtained as a colorless oil (0.56 g, 78% yield). R_(f) ≅0.36(ethyl acetate/cyclohexane 1:1).

Step B: 3-(1-Naphthyl)-L-alanine-N,O-dimethyl hydroxamate

A solution ofN-tert-butoxycarbonyl-[3-(1-naphthyl)-L-alanine]-N,O-dimethylhydroxamate (0.560 g, 1.5 mmol) in formic acid (20 ml) is stirred atroom temperature for 4 hours. The solvent is removed invacuo. Theresidue is taken off in ethyl acetate (50 ml) and washed three timeswith a saturated solution of sodium carbonate (3×10 ml) and dried overanhydrous magnesium sulphate. Filtration and evaporation of the solventinvacuo affords a colorless oil which is used without purification inthe next step (0.330 g, 85% yield).

Step C:N-Benzyloxycarbonyl-L-valyl-3-(1-naphthyl)-L-alanine-N,O-dimethylhydroxamate

To a solution of Z-L-valine anhydride (0.581 g, 1.2 mmol) in anhydrousdichloromethane (10 ml) is added 3-(1-naphthyl)-L-alanine-N,O-dimethylhydroxamate (0.320 g, 1.2 mmol). The mixture is stirred at roomtemperature overnight. The solvent is removed invacuo and the residue ispurified by flash chromatography (silica gel: 3:7 ethylacetate/cyclohexane). The title compound is obtained as a white solid(0.470 g, 80% yield). R_(f) ≅0.23 (ethyl acetate/cyclohexane).

Step D: N-Benzyloxycarbonyl-L-valyl-3-(1-naphthyl)-L-alaninal

To a solution of N-benzyloxycarbonyl-L-valyl-3-(1-naphthl)-L-alanine-N,O-dimethyl hydroxamate (0.470 g, 0.96 mmol) in anhydrousdiethyl ether (10 ml) and anhydrous THF (10 ml) at 0° C. is addedlithium aluminum hydride (0.042 g, 1.1 mmol). The reaction is stirred at0° C. for 1 hour. A solution of 1M potassium hydrogenosuphate (5 ml) isthen added. The mixture is extracted three times with ethyl acetate(3×30 ml). The organic layer is washed with 1N hydrochloric acid, waterand brine, and dried over anhydrous magnesium sulphate. Filtration andevaporation of the solvent invacuo affords a white solid which ispurified by recrystallization in ethylacetate/pentane to give the titlecompound (0.260 g, 63% yield). R_(f) ≅0.36 (Ethyl acetate/cyclohexane1:1) MS: [MH]⁺ =433 [MNH₄ ]⁺ =450 Analysis calculated for C₂₆ H₂₈ N₂ O₄: Calc: C, 72.20; H, 6.52; N, 6.48. Found: C, 71.87; H, 6.50; N, 6.48.

EXAMPLE 20 ##STR57## Preparation ofN-Benzyloxycarbonyl-L-valyl-4-(phenyl-carbonylamino)-L-phenylalaninal

Step A: N-Benzyloxycarbonyl-L-valyl-4-nitro-L-phenylalanine methyl ester

To a solution of Z-L-valine anhydride (4.80 g, 10 mmol) in anhydrousdichloromethane (50 ml) is added 4-nitro-L-phenylalanine methyl ester(2.24 g, 10 mmol). The mixture is stirred at room temperature overnight.The solvent is removed invacuo and the residue is purified by flashchromatography (silica gel: 4:6 ethyl acetate/cyclohexane) to give thetitle compound (2.10 g, 56% yield). R_(f) ≅0.32 (ethylacetate/cyclohexane 1:1).

Step B:N-Benzyloxycarbonyl-L-valyl-4-(phenylcarbonyl-amino)-L-phenylalaninemethyl ester

A solution of N-benzyloxycarbonyl-L-valyl-4-nitro-L-phenylaninephenylalanine methyl ester (0.91 g, 2 mmol) and Tin (II) chloridedihydrate (1.56 g, 7 mmol) in absolute ethanol (50 ml) andN,N-dimethylformamide (5 ml) is heated under reflux for 4 hours. Themixture is cooled and diluted with water, neutralized with sodiumhydrogenocarbonate, extracted three times with ethyl acetate (3×50 ml).The organic layer is dried over magnesium sulphate. After filtration andevaporation of the solvent, the residue is taken up in anhydrousdichloromethane (20 ml) and cooled to 0° C. Triethylamine (0.202 g, 2mmol), followed by benzoyl chloride (0.281 g, 2 mmol) are added. Themixture is stirred at room temperature overnight. The solvent is removedinuacuo and the residue purified by flash chromatography (silica gel:98;2 dichloromethane/methanol) to give the title compound (0.500 g, 50%yield).

Step C:N-Benzyloxycarbonyl-L-valyl-4-(phenylcarbonyl-amino)-L-phenylalanine

To a solution ofN-benzyloxycarbonyl-L-valyl-4-(phenylcarbonylamino)-L-phenylalaninemethyl ester (0.500 g, 0.94 mmol) in dioxane (30 ml) is added lithiumhydroxide monohydrate (0.084 g, 2 mmol) in water (10 ml). The reactionis stirred at room temperature overnight. The mixture is taken off inwater (20 ml) and washed twice with ethyl acetate (2×20 ml). The aqueousphase is acidified until pH ˜2 with 1N hydrochloric acid and extractedthree times with ethyl acetate (3×20 ml). The organic layer is driedover anhydrous magnesium sulphate. After filtration and remtial of thesolvent invacuo the title compound is obtained as a white solid (0.400g, 82% yield). MS: [MH]⁺ =518 [MNH₄ ]⁺ =535.

Step D:N-Benzyloxycarbonyl-L-valyl-4-(phenylcarbonyl-amino)-L-phenylalanine-N,O-dimethylhydroxamate

To a solution ofN-benzyloxycarbonyl-L-valyl-4-(phenylcarbonylamino)-L-phenylalanine(0.390 g, 0.75 mmol) in anhydrous N,N-dimethylformamide (5 ml) andanhydrous dichloromethane (15 ml) at 0° C., are addedN,N-dicyclohexyl-carbodiimide (0.155 g, 0.75 mmol) andhydroxybenzotriazole (0.115 g, 0.75 mmol). After 10 minutes stirringN,O-dimethyl hydroxamate chlorohydrate (0.073 g, 0.75 mmol) andN-methylmorpholine (0.076 g, 0.75 mmol) are added. The reaction isstirred at room temperature overnight, the solvent removed in vacuo, andthe residue is taken up in ethyl acetate and filtered. The filtrate isconcentrated and the residue is purified by flash chromatography (silicagel: 98:2 dichloromethane/methanol) to give the title compound (0.230 g,53% yield).

R_(f) ≅0.59 (CH₂ Cl₂ /MeOH 9:1) MS: [MH]⁺ =561 [MNH₄ ]⁺ =578.

Step E:N-Benzyloxycarbonyl-L-valyl-4-(phenylcarbonyl-amino)-L-phenylalaninal

To a solution ofN-benzyloxycarbonyl-L-valyl-4-(phenylcarbonylamino)-L-phenylalanine-N,O-dimethylhydroxamate (0.230 g, 0.41 mmol) in anhydrous diethyl ether (10 ml) andanhydrous THF (5 ml) at 0° C., is added lithium aluminum hydride (0.017g, 0.45 mmol). The reaction is stirred at 0° C. for 1 hour. A 1Msolution of potassium hydrogenosulphate (5 ml) is added and the mixtureis extracted three times with ethyl acetate (3×15 ml). The organic layeris washed with 1N hydrochloric acid, water and brine, and then driedover anhydrous magnesium sulphate. After removal of the solvent invacuo,the residue is purified by flash chromatography (silica gel: 98:2dichloromethane/methanol) to give the title compound (0.050 g, 25%yield).

R_(f) ≅0.43 (CH₂ Cl₂ /MeOH 9:1) MS: [MH]⁺ =502 [MNH₄ ]⁺ =519.

EXAMPLE 21 ##STR58## Preparation of2(D)-[3-(4-Benzyloxy-phenyl)-2-formyl-propionylamino]-3-methyl-N-phenethylbutyramide

Step A: 4-Benzyloxybenzyl malonic acid, tert-butyl, ethyl ester

To a suspension of 45% sodium hydride (6.40 g, 0.12 mmol) in anhydroustetrahydrofuran (100 ml) under nitrogen, is added tert-butyl ethylmalonate (20,7 g, 0.11 mmol) in anhydrous tetrahydrofuran (100 ml). Themixture is stirred at room temperature for 2 hours. 4-benzyloxybenzylbromide (29.60 g, 0.11 mmol) in anhydrous THF (50 ml) is then added. Thereaction is stirred at room temperature overnight, hydrolized with waterand concentrated. The mixture is extracted three times with diethylether (3×200 ml). The residue is purified by flash chromatography(silica gel: 9:1 toluene/ ether) and recrystallized (diethyl ether:pentane) to give the title compound (20.0 g; 47% yield).

R_(f) =0.65 (toluene: ether 9:1).

Step B: 4-Benzyloxybenzyl malonic acid, ethyl ester

A solution of 4-Benzyloxybenzyl malonic acid, tert-butyl-ethyl ester(19.0 g, 49.5 mmol) in trifluoroacetic acid (200 ml) is stirred at 0° C.for one hour. The solvent is removed in vacuo. The residue isrecrystallized in diethyl ether/pentane to give the title compound (8.50g; 53% yield).

Step C: 2(D)-[3-(4-Benzyloxy-phenyl)-2-carboxydiethylester-propionylamino]-3-methyl-N-phenethyl butyramide

To a solution of 4-Benzyloxybenzyl malonic acid, ethyl ester (8.50 g,25.9 mmol) in anhydrous dichloromethane (150 ml) at 0° C., are addedN,N-dicyclohexylcarbodiimide (5.33 g, 25.9 mmol), hydroxybenzotriazole(3.96 g, 25.9 mmol) and (D) valine phenethylamide (5.70 g, 25.9 mmol).The reaction is stirred at room temperature overnight.

The precipitate is filtered and the filtrate is concentrated. Theresidue is purified by flash chromatography (silica gel: 3:7 ethylacetate/cyclohexane) to give the title compound (7.0 g; 51% yield) as awhite solid.

R_(f) ≅0.33 (ethyl acetate/cyclohexane 1:1) MS: [MH]⁺ =531 Analysiscalculated for C₃₂ H₃₈ N₂ O₅ : Calc: C, 72.43; H, 7.22; N, 5.28. Found:C, 72.15; H, 7.36; N, 5.39.

Step D:2(D)-[3-(4-Benzyloxy-phenyl)-2-carboxy-propionylamino]-3-methyl-N-phenethylbutyramide

To a solution of2(D)-[3-(4-Benzyloxy-phenyl)-2-carboxydiethylester-propionylamino]-3-methyl-N-phenethylbutyramide (7.0 g, 13.2 mmol) in 2-methoxy ethanol (100 ml) is added asolution of lithium hydroxide (0.84 g, 20 mmol) in water (50 ml). Thereaction is refluxed for 12 hours. The solvent is removed invacuo, theresidue taken off in water (100 ml) and washed two times with ethylacetate (2×30 ml). The aqueous phase is acidified until pH ˜2 with 3Nhydrochloric acid, saturated with sodium chloride and extracted threetimes with ethyl acetate (3×50 ml). The organic layer is dried overanhydrous magnesium sulfate.

After filtration and removal of the solvent invacuo, the residue ispurified by recrystallization (ethyl acetate/pentane) to give the titlecompound (4.0 g; 60% yield).

MS: [MH]⁺ =503

Step E: 2(D)-[3-(4-Benzyloxy-phenyl)-2-N,O-dimethylcarboxamate-propionylamino]-3-methyl-N-phenethyl butyramide

To a solution of2(D)-[3-(4-Benzyloxy-phenyl)-2-carboxy-propionylamino]3-3-methyl-N-phenethylbutyramide (4.00 g, 8 mmol) in anhydrous dichloromethane (100 ml) andN,N dimethylformamide (10 ml) at 0° C. are added N,Odimethylhydroxamate, hydrochloride (0.78 g, 8 mmol) N-methylmorpholine(0.81 g, 8 mmol) and N,N dicyclohexylcarbodiimide (1.65 g, 8 mmol). Thereaction is stirred at room temperature overnight. The mixture isfiltered and the filtrate concentrated invacuo. The residue is purifiedby flash chromatography (silica gel: 1:1 ethyl acetate/cyclohexane) togive the title compound as a white solid (3.20 g; 74% yield).

R_(f) ≅0.45 (ethyl acetate) MS: [MH]⁺ =546 [MNH₄ ]⁺ =563 Analysiscalculated for C₃₂ H₃₉ N₃ O₅ : Calc: C, 70.44; H, 7.20; N, 7.70. Found:C, 70.22; H, 7.02; N, 7.65.

Step F:2(D)-[3-(4-Benzyloxy-phenyl)-2-formyl-propionyl-amino]-3-methyl-N-phenethylbutyramide

To a solution of 2(D)-[3-(4-Benzyloxy-phenyl)-2-N,O-dimethylcarboxamate-propionylamino]-3-methyl-N-phenethyl butyramide (3.0 g, 5.5mmol) in anhydrous tetrahydrofuran (50 ml is added lithium aluminumhydride (0.240 g, 6.3 mmol). The reaction is stirred at 0° C. for onehour then hydrolized with a solution 1M of potassium hydrogeno sulfate(20 ml) and extracted three times with ethyl acetate (3×50 ml). Theorganic layer is washed with 1N hydrochloric acid, water and brine thendried over anhydrous magnesium sulfate. Filtration and removal of thesolvent invacuo affords a white solid which is recrystallized in ethylacetate/pentane to give the title compound (2.30 g; 86% yield).

MS: [MH]⁺ =487 [MNH₄ ]⁺ =504 Analysis calculated for C₃₀ H₃₄ N₂ O₄,0.5H₂ O: Calc.: C, 72.70; H, 7.12; N, 5.65. Found: C, 72.90; H, 7.03; N,5.89.

EXAMPLE 22 ##STR59## Preparation ofN-Benzyloxycarbonyl-L-valyl-4-nitro-L-phenylalaninal

To a solution of N-Benzyloxycarbonyl-L-valyl-4-nitro-L-phenylalaninalmethyl ester (0.375 g, 0.8 mmol) in anhydrous toluene (10 ml), at -78°C. under nitrogen is added a solution of 1.2 M diisobutyl aluminumhydride in hexane (3 ml, 3.5 mmol). The reaction is stirred at -78° C.for one hour, then hydrolized with a saturated solution of potassiumsodium tartrate. The temperature is allowed to rise to room temperature.The mixture is acidified until pH ˜3 with a 1M solution of potassiumhydrogenosulfate and extracted three times with ethyl acetate (3×20 ml).The organic layer is dried over anhydrous magnesium sulfate. Filtrationand removal of the solvent invacuo affords a residue which is purifiedby flash chromatography (silica gel: 1:1 ethyl acetate/cyclohexane). Thetitle compound is obtained with 20% yield (70 mg).

R_(f) ≅0.50 (ethyl acetate).

EXAMPLE 23 ##STR60## Preparation of4-D-(N-Benzyloxycarbonyl)amino-2,2-difluoro-3-oxo-5-phenyl-N-benzylpentanamide

Step A: N-Benzyloxycarbonyl-D-phenylalanine,N,O-dimethyl hydroxamate

To a solution of Z(D)Phe-OH (31 g, 0.103 mol) in anhydrous methylenechloride (300 mL) at 0° C. are added N,N'-dicyclohexyl carbodiimide(21.21 g, 0.103 mol) and hydroxybenzotriazole hydrate (15.76 g, 0.103mol). Stir at 0° C. for 10 minutes, add N,O-dimethylhydroxylamine, HCl(10.04 g, 0.103 mol) and N-methyl morpholine (10.40 g, 0.103 mol) to thereaction and stir at 25° C. for 12 hours.

Filter the mixture, wash with methylene chloride and concentrate thefiltrate under vacuo to provide the crude 30 hydroxamate as an oil.Purify the crude residue by flash chromatography (silica gel: ethylacetate/cyclohexane 4:6) to provide the title compound as a colorlessoil (29.8 g, 85% yield).

R_(f) ≅0.37 (ethylacetate:cyclohexane 1:1).

Step B: N-Benzyloxycarbonyl-D-phenylalaninal

Lithium aluminum hydride (3.8 g, 0.1 mol) is added to a solution of thehydroxamate (29 g, 0.084 mol) in anhydrous diethyl ether (500 mL) at 0°C. under an inert atmosphere. Stir for 1 hour. Add cautiously a 1Msolution of potassium hydrogenosulphate (150 mL). Separate the phasesand extract the aqueous layer twice with diethylether (2×150 mL). Washthe combined organic layers with an aqueous 3N hydrochloric acid (50mL), water (50 mL) and brine (50 mL). Dry over anhydrous magnesiumsulphate, filter and concentrate under vacuum to provide the titlecompound. Purify by recrystallization in ethyl acetate/pentane (10.9 g,46% yield).

R_(f) ≅0.41 (ethylacetate:cyclohexane 1:1).

Step C: 4-D-N-Benzyloxycarbonylamino-2,2-difluoro-3-hydroxy-5-phenylpentanoic acid, ethyl ester

To a suspension of zinc (2.30 g, 35.2 mAtq) in anhydrous tetrahydrofuran(15 mL) under nitrogen, add a mixture of ethylbromodifluoroacetate (7.14g, 35.2 mmol) and N-benzyloxycarbonyl-(D)-phenylalaninal (4.75 g, 16.8mmol) in anhydrous tetrahydrofuran (30 mL). After addition of 2 mL ofthis solution, heat the suspension at reflux with stirring. Maintaingentle reflux by slow addition (dropwise) of the rest of the solution ofaldehyde and bromoester. Stir the mixture for 4 hours at roomtemperature after completion of the addition. Hydrolyze by addition of1M potassium hydrogenosulphate (30 mL). Extract the solution with ethylacetate (3×30 mL). Wash the combined organic layers with brine and dryover anhydrous magnesium sulphate. Filter and concentrate the filtrateunder vacuum. Purify the crude residue by flash chromato-graphy (silicagel, ethyl acetate:cyclohexane 1:9) to provide the title compound as awhite solid (2.95 g, 43% yield).

R_(f) ≅0.50 (ethylacetate:cyclohexane 1:1). Anal. Calcd for C₂₁ H₂₃ NO₅F₂ : C, 61.91; H, 5.69; N, 3.44. Found: C, 62.19; H, 5.75; N, 3.55.

Step D:4-D-N-Benzyloxycarbonylamino-2,2-difluoro-3-hydroxy-5-phenyl-N-benzylpentanamide

Stir a solution of the ester of Example 23, Step C (1.42 g, 3.5 mmol)and benzylamine (1.93 g, 18 mmol) in anhydrous tetrahydrofuran (10 mL)at 25° C. for 12 hours. Take off with ethyl acetate (100 mL) and washthree times with 1N aqueous hydrochloric acid (3×15 mL), water (15 mL)and brine (15 mL). Dry over anhydrous magnesium sulphate, filter andconcentrate the filtrate. Purify the crude residue by flashchromatography (silica gel, gradient ethyl acetate:cyclohexane 3:7 toethyl acetate). The title compound is obtained as a white solid (1.16 g,71% yield).

R_(f) ≅0.40 (ethylacetate:cyclohexane 1:1) MS: MH⁺ =469.

Step E:4-D-(N-Benzyloxycarbonyl)amino-2,2-difluoro-3-oxo-5-phenyl-N-benzylpentanamide

To a mixture of Dess-Martin periodinane reagent (0.36 g, 0.85 mmol) indry methylene chloride, add a solution of the alcohol of Example 23,Step D (0.11 g, 0.23 mmol) in methylene chloride and N,N-dimethylformamide (2 mL, 1 mL), and stir at 25° C. for 3 hours. Evaporate thesolvent under vacuum, and purify the crude residue by flashchromatography (silica gel, ethyl acetate:cyclohexane 1:1) followed byrecrystallization (ethyl acetate/pentane). The title compound isobtained as a white solid (0.074 g, 69% yield).

R_(f) ≅0.45 (ethylacetate:cyclohexane 1:1). Anal. Calcd for C₂₆ H₂₄ N₂O₄ F₂.0.5 H₂ O: C, 65.68; H, 5.30; N, 5.89 Found: C, 66.05; H, 5.22; N,5.97. MS: MH⁺ =467 MP: 130° C.

EXAMPLE 24 ##STR61## Preparation of4-(N-Benzyloxycarbonyl-L-norvalyl)amino-2,2-difluoro-3-oxo-5-phenyl-N-benzylpentanamide

Step A:4-(N-Benzyloxycarbonyl-L-norvalyl)amino-2,2-difluoro-3-hydroxy-5-phenyl-N-benzylpentanamide

The title compound is obtained from the amine described in Example 7,Step D, and carbobenzoxy-L-norvaline following the coupling proceduredescribed in Example 8, Step C (65% yield).

R_(f) ≅0.40 (ethylacetate:cyclohexane 1:1) MS: MH⁺ =568.

Step B:4-(N-Benzyloxy-L-norvalyl)amino-2,2-difluoro-3-oxo-5-phenyl-N-benzylpentanamide

To a suspension of Dess-Martin periodinane (0.271 g, 0.64 mmol) inmethylene chloride (5 mL) add the alcohol described in Example 23, StepA (0.09 g, 0.16 mmol) in 5 mL dichloromethane. Stir at 25° C. for 4hours. Concentrate under vacuum and purify the crude residue by flashchromatography (silica gel, ethyl acetate:cyclohexane 2:8) followed byrecrystallization (ethyl acetate/pentane). The title compound isobtained as a white solid (0.04 g, 45% yield).

R_(f) ≅0.41 (ethylacetate:cyclohexane 1:1) MS: MH⁺ =566 Anal. Calcd forC₃₁ H₃₃ N₃ O₅ F₂.0.5 H₂ O: C, 64.80; H, 5.96; N, 7.31 Found: C, 64.82;H, 5.92; N, 7.16.

EXAMPLE 25 ##STR62## Preparation of4-(N-Benzyloxycarbonly-L-tert-leucyl)amino-2,2-difluoro-3-oxo-5-phenyl-N-trimethvlsilylmethylpentanamide

Step A:4-(N-Benzyloxycarbonyl-L-tert-leucyl)amino-2,2-difluoro-3-hydroxy-5-phenyl-N-trimethylsilylmethylpentanamide

A mixture of the amine described in Example 8, Step B [prepared as itstrifluoroacetic acid salt (0.222 g, 0.5 mmol)], N-methyl morpholine (115μL, 1.05 mmol) and carbobenzoxy-L-tert-leucyl anhydride [previouslyprepared in Situ from the acid and N,N'-dicyclohexyl carbodiimide in 3mL of dimethylformamide (0.566 g, 1.1 mmol)] is kept overnight understirring at room temperature. The mixture is diluted with ethyl acetate,washed twice with water and the aqueous layer concentrated under vacuum.The crude residue is purified by flash chromatography (silica gel, ethylacetate:petroleum ether 3:7) to give the title alcohol in 52% yield(0.15 g)

R_(f) ≅0.69 (ethylacetate:petroleum ether 4:6)

Step B:4-(N-Benzyloxycarbonyl-L-tert-leucyl)amino-2,2-difluoro-3-oxo-5-phenyl-N-trimethylsilylmethylpentanamide

The title ketone is prepared in 69% yield from the alcohol of Example25, Step A, using the Swern oxidation procedures described in Example 8,Step D.

R_(f) ≅0.30 (ethylacetate:petroleum ether 3:7).

Anal. Calcd for C₂₉ H₃₉ F₂ N₃ O₅ Si, 0.5 H₂ O: C, 59.57; H, 6.90; N,7.19 Found: C, 59.65; H, 6.89; N, 7.00.

EXAMPLE 26 ##STR63##

Preparation of6-(N-Benzyloxycarbonyl-L-valyl)amino-4,4-difluoro-1-phenyl-7-methyl-3,5-dioxooctane

Step A: Benzyloxycarbonyl-L-valyl-valinal

The title aldehyde is prepared in 43% yieldfrom-N-benzyl-oxycarbonyl-L-valyl-L-valine, ethyl ester using thereduction method described in Example 12, Step D.

R_(f) ≅0.25 (silica gel ethylacetate:petroleum ether 3:7) MS: MH⁺ =335,MNH₄ ⁺ =352.

Step B:6-(N-Benzyloxycarbonyl-L-valyl)amino-4,4-difluoro-1-phenyl-7-methyl-5-hydroxy-3-oxooctane

The title difluoro alcohol is obtained in 39% yield from the aldehyde ofExample 26, Step A, and 1-chloro-1,1-difluoro-2-oxo-4-phenylbutanefollowing the procedure described in Example 6, Step C.

R_(f) ≅0.43 (silica gel ethylacetate:petroleum ether 3:7) MS: MH⁺ =519,MNH₄ ⁺ =536.

Anal. Calcd for C₂₈ H₃₆ F₂ N₂ O₅ : C, 64.85; H, 7.00; N, 5.40 Found: C,65.11; H, 7.25; N, 5.22.

Step C:6-(N-Benzyloxycarbonyl-L-valyl)amino-4,4-difluoro-1-phenyl-7-methyl-3,5-dioxooctane

The title diketone is obtained from the alcohol of Example 26, Step C,using the Swern oxidation described in Example 8, Step D (34% yield).

R_(f) ≅0.31 (silica gel ethylacetate:petroleum ether 2:8) MS: MH⁺ =517,MNH₄ ⁺ =534.

Anal. Calcd for C₂₈ H₃₄ F₂ N₂ O₅ : C, 65.10; H, 6.63; N, 5.42 Found: C,65.22; H, 6.90; N, 5.05.

EXAMPLE 27 ##STR64## Preparation of(1-Phenethyl-2-oxy)carbonyl-L-valyl-phenylalaninal Step A:(1-Phenethyl-2-oxy)carbonyl-L-valine, methyl ester

To a suspension of carbonyldiimidazole (4.02 g, 24.8 mmol) in anhydrousdichloromethane (10 mL) is added valine methyl ester (1.63 g, 12.4 mmol)in dichloromethane (3 mL) dropwise via a syringe (over a 3 minuteperiod). The reaction mixture becomes homogeneous and is continued tostir at room temperature for 15 minutes. The mixture is thenconcentrated invacuo to give a white solid. The solid is suspended inanhydrous toluene (10 mL) and 2-phenyl-ethanol (7.4 mL, 62.0 mmol) isadded dropwise. The reaction mixture becomes clear and is heated at 68°C. (oil bath) for 3 hours. The solvent is removed by rotoevaporation andthe residue is taken up in dichloromethane, washed with water twice andbrine, dried over magnesium sulphate, filtered, and concentratedinvacuo. The crude material is purified on silica gel withhexane:petroleum ether (80:20 to 60:40) as eluent to give 2.51 g (72%)of the desired product.

Step B: (1-Phenethyl-2-oxy)carbonyl-L-valine

To a mixture of the carbamate of Example 27, Step A (0.43 g, 1.54 mmol)in 5 mL of methanol and 1 mL of water is added lithium hydroxide (1.0 M,1.4 mL, 1.4 mmol). The reaction is heated to reflux (80° C.) for 3 hoursand stirred at room temperature overnight. More lithium hydroxide (1M,0.5 mL) is added and the reaction is continued to stir at roomtemperature for another 3 hours. The reaction mixture is concentratedinvacuo. Dichloromethane and water are added. The two layers areseparated, and the aqueous layer is acidified to pH 1 with HCl (1M) andextracted with dichloromethane twice. The combined organic layers aredried over magnesium sulphate, filtered, and concentrated in vacuo toyield 0.33 g (81%) of the desired acid.

Step C: (1-Phenethyl-2-oxy)carbonyl-L-valyl-L-phenyl-alaninol

To a solution of the acid of Example 27, Step C (0.33 g, 0.88 mmol),phenylalaninol (0.3 g, 1.24 mmol), 1-hydroxybenzotriazole (0.27 g, 1.24mmol), N-methyl morpholine (0.22 mL, 1.24 mmol) in dichloromethane (10mL) is added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (0.33 g, 1.24mmol). The reaction mixture is stirred at room temperature overnight,diluted with dichloromethane, washed with iN HCl, water, saturatedaqueous sodium bicarbonate, water and brine, dried over magnesiumsulphate, filtered and concentrated invacuo. The remaining white fluffysolid is crystallized from hexane/ethyl acetate to yield the titlecompound as a white solid (0.35 g, 71%)

Step D: (1-Phenethyl-2-oxy)carbonyl-L-valyl-phenylalaninal

The title aldehyde is obtained in 54% yield from the alcohol of Example27, Step C, using the oxidation method described in Example 8, Step D.

Anal. Calcd for C₂₃ H₂₈ N₂ O₄ : C, 69.67; H, 7.12; N, 7.07 Found: C,69.61; H, 7.22; N, 6.77.

EXAMPLE 28 ##STR65## Preparation of4-(N-Benzyloxycarbonyl-L-tert-leucyl)amino-2,2-difluoro-3-oxo-5-phenyl-N-benzylpentanamide

Step A:4-(N-Benzyloxycarbonyl-L-tert-leucyl)amino-2,2-difluoro-3-hydroxy-5-phenyl-N-benzylpentanamide

The title compound is obtained from the amine depicted in Example 7,Step D, and carbobenzoxy-L-tert-leucine using a coupling procedureanalogous to that described in Example 25, Step A (77% yield).

R_(f) ≅0.37 (silica gel ethylacetate:petroleum ether 4:6).

Step B:4-(N-Benzyloxycarbonyl-L-tert-leucyl)amino-2,2-difluoro-3-oxo-5-phenyl-N-benzylpentanamide

The title final compound is prepared in 85% yield from the alcohol ofExample 28, Step A, using the Swern oxidation procedure analogous tothat described in Example 8, Step D. R_(f) ≅0.24 (silica gelethylacetate:petroleum ether 3:7).

Anal. Calcd for C₃₂ H₃₅ F₂ N₃ O₅ : C, 66.31; H, 6.03; N, 7.25 Found: C,65.99; H, 6.15; N, 7.13.

EXAMPLE 29

The activity of the compounds of this invention to prevent or reduce theaccumulation of β-amyloid plaques and thus the usefulness in thetreatment of senile dementia of the Alzheimer's type and otherconditions known to be associated with the formation of β-amyloid plaquesuch as Down's syndrome can be demonstrated by various invitro and invivo models of β-amyloid plaque formation. For example the ability ofthe compounds of this invention to prevent or reduce the accumulation ofβ-amyloid plaques can be demonstrated by several cellular and cell freein vitro methods described as Assay's 1-3 as follows. These assays makeuse of the fact that native β-APP is expressed by all cells and isprocessed to produce 11-12 KDa C-terminal fragments and β-amyloid. Theendogenous level of β-APP expression can be enhanced if desired bytransfecting β-APP cDNA sequences, e.g., β-APP (751) into the cellsusing standard methodology.

IN VITRO ASSAYS

Assay #1: Immunoprecipitation

Cells; CHO-Kl (Chinese Hamster Ovary; ATCC origin) cell line stablytransfected to express large amounts of βAPP-695, and referred to as"CP-6-36" are used for screening inhibitors. Other mammalian culturedcell lines can also be used and have been used. For example, the humanneuronal cell line SK-N-MC (ATCC origin) gives good results under thesame assay conditions. Transfection with βAPP-695 is not a requisite ofβA4 production; it merely enhances the βA4 signal. In preparation for anexperiment, CP-6-36 cells are seeded at low density in 10 cm dishes andgrown for two to four days to a confluent monolayer (˜1.5×10⁷ cells perdish) in a 37° C./5% CO₂ incubator; growth media consists of DMEM21/Coon's F12 (1:1) +10% FBS (fetal bovine serum) +50 U/mL penicillinand 50 μg/mL streptomycin.

Treatment: All compounds are initially screened on CP-6-36 cells at adose of 200 μM. Prior to testing, a 20 mM stock of each compound to betested is prepared using cell culture grade DMSO as a solvent. Each 20mM stock compound is then diluted 100-fold into serum free EMEM mediadeficient in the amino acids cysteine and methionine ("Cys-/Met- EMEM"),giving a 200 μM final concentration of compound in the media. To beginthe experiment the cells are "starved" for cysteine and methionine bywashing the cell monolayers 3 times with 3 mL/dish of Cys-/Met- EMEM,then incubating (37° C./5% CO₂) with 3 mL/dish of the same media for 15minutes. This media is aspirated from the dishes, then media containingthe compounds at 200 μM is added at 3 mL/dish. These plates and a"control" dish (3 mL/dish Cys-/Met- EMEM containing 1% DMSO and nocompound) are incubated as above for 15 minutes. This media is aspirated, then to each dish an additional 3 mL of the media from the previousstep now containing ³⁵ S-Trans label (³⁵ -S labeled cysteine andmethionine) at ˜150 μCi/mL is added. The cells are incubated as abovefor 4 hours.

Harvest: At the end of the 4 hour labeling period, the cells areobserved under the microscope for overall appearance and to check forgross toxicity effects of the compounds, after which the dishes of cellsare placed on ice. The conditioned media from each dish is transferredto 15 mL conical screw-cap tubes, centrifuged at 2000 rpm for 10 minutesand transferred to a set of similar tubes, leaving behind any pelletedcells. The labeled cell mono-layers are washed three times with 2mL/dish phosphate-buffered saline (PBS), then 1 mL of a buffer whichpromotes cell lysis (5% Triton X-114; 20 mM Tris, pH 7.5; 300 mM NaCl;protease inhibitors) is added to each dish, followed by a 10 minuteincubation on ice. The cell lysates are scraped from the dishes andtransferred to 1.5 mL microfuge tubes. The lysates are then sonicatedfor 4 minutes on ice, spun at high speed in a microfuge for 10 minutes,then transferred to 15 mL conical screw-cap tubes, leaving behind thepellet of cell debris.

Immunoprecipitation: In preparation for immuno-precipitation, thelysates harvested above are diluted in 5 mL of 1×RIPA buffer (10 mMTris, pH 8.0; 150 mM NaCl; 0.125% NaN₃ ; 1% Triton X-100; 1%deoxycholate; 0.1 SDS); the conditioned media samples areimmunoprecipitated without dilution. Both conditioned media and lysatesare first precleared by adding 5 μL of normal rabbit serum to eachsample, rocking 10 minutes at room temperature, followed by the additionof 100 μL 10% protein A-Sepharose (PAS) in RIPA buffer, and rocking atroom temperature for 1.5 hours. The samples are then centrifuged at 3000rpm, and the supernatants are transferred to new 15 mL tubes. Theprecleared lysates are then immunoprecipitated by adding 30 μL of anantibody which recognizes the carboxyl terminus of βSAPP to each tube,rocking for 10 minutes at room temperature, followed by the addition of100 μL of 10% PAS and rocking at room temperature for 1.5 hours. Theprecleared conditioned media samples are immunoprecipitated identically,however 45 μL of an antibody which recognizes βA4 is used instead of thecarboxyl terminal directed antibody. All samples are then centrifugedfor 1 minute at 3000 rpm to pellet the PAS-antibody complexes, and theresulting pellets are washed extensively; 4 times with a high saltbuffer (50 mM Tris, pH 7.5; 500 mM NaCl; 5 mM EDTA; 0.5% Nonidet P-40),3 times with a low salt buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 5 mMEDTA; 0.5 Nonidet P-40), and 2 times with 10 mM Tris buffer, pH 7.5.

Gel electrophoresis: The washed pellets are boiled for 5 minutes in 50μL of 2×Laemmli gel loading buffer. These samples as well as molecularweight markers are loaded onto a 16.5% SDS-polyacrylamide gel withTris/Tricine reservoir buffers. The gel is run at 90V for ˜18-20 hours,fixed in 20% methanol/20% acetic acid, and dried onto filter paper at65° C. for 2 hours. Autoradiography is used to visualize the results.

Analysis: Results are obtained by analysis of the auto-radiogram. Apositive acting compound is one which inhibits the 4 kDa βA4 proteinband relative to the control sample, and additionally some may increaselevels of the 9-12 kDa C-terminal protein bands relative to the controlsample. Quantitation of inhibition of βA4 or increase of C-terminalbands can be made by densitometric scanning of the bands, normalized tocontrol bands. A negative acting compound is one which shows no changein the yield of 4 kDa βA4 or 9-12 kDa C-terminal protein bands, relativeto the bands from the control sample.

Additional testing: If a compound is found to be active (i.e.,substantial inhibition of 4 kDa βA4 with concomitant increase inC-terminal fragments, by gel analysis), then a dose response experimentis performed to determine the lowest dose of compound necessary toelicit above effects. The dose range typically used is 12.5-300 μM, andwith the exception of these dose changes, the experiment is doneidentically as described above. If a compound is found to be onlyslightly active or not active at all, the experiment is repeated using ahigher dose, typically 400 μM. If a compound is found to be toxic (i.e.,cells appear unhealthy by observation under the microscope, or lysatesappear to not have been labeled well after gel analysis), then thecompound is tested again at lower doses, for example: 25, 50 and 100 μM,to determine the effect of the compound at a non-toxic dose.

Assay #2: Radioimmunoassay

Preparation and Speak concentration of media for the RIA: Culturedmammalian cells such as Chinese hamster ovary (CHO) cells or humanneuronal SK-N-MC cells produce β-amyloid and secrete this peptide intothe culture medium. If cells are treated with potential inhibitors ofβ-amyloid formation, no soluble β-amyloid would be found in the mediumof the treated cells. As with Assay #1, varying doses of inhibitorycompounds can be tested beginning with 200 μM. For CHO cells, both wildtype and β-APP695 transfected, 10 cm plates are incubated in 2 mL EMEM(serum free) for 4 to 6 hours at 37° C. in the presence or absence ofinhibitory compounds to be evaluated. The medium is removed andcentrifuged for 10 minutes at 1500 rpm (Sorvall RT6000B) to remove anycells/debris. The medium is either used immediately or stored at -20° C.The Sepak C18 step is performed to remove salts and other unwantedcontaminants and to concentrate the β-amyloid peptides. Medium sample (2ml) is passed through a C18 Sepak cartridge and the cartridge is washedin 2 ml 5% CH₃ CN in 0.1% TFA. The runthrough and the 5% CH₃ CN wash arediscarded. The cartridge is eluted wit h 2 mL 25% CH₃ CN in 0.1% TFAfollowed by 2 mL elution in 50% CH₃ CN in 0.1% TFA. Both elutions arecollected and dried in the speedvac and taken up in 125 μL to 250 μL of10% isopropanol in water for assaying in the RIA. The 25% CH₃ CNfraction contains most of the phenol red from the media but no β-amyloidpeptide. The 50% CH₃ CN fraction contains the β-amyloid peptides.

Preparation and HPLC purification of ¹²⁵ I labeled β-amyloid 1-40:Synthetic β-amyloid 1-40 (10 μg) is labeled with ¹²⁵ I (lmci) by theChloramine T method. The reaction is carried out at room temperature. Inan eppendorf tube, 10 μL of ¹²⁵ I (lmci in NAOH solution) is added to 10μL of β-amyloid 1-40 (lmg/mL in 20% Isopropanol) and 80 μL 0.1MNaPhosphate, pH 7.4 and mixed. The reaction is initiated by adding 30 μLChloramine-T (lmg/mL, in 0.1M NaPhosphate, pH 7.4) mixing and incubating1 minute. The reaction is stopped by adding 150 μL NaMetabisulfite (2mg/mL, 0.1M NaPhosphate, pH 7.4).

The reaction mixture (280 μL) is diluted with equal volume of water andrun on a Sepak C18 cartridge to separate the labeled peptide. The Sepakis washed twice in 5% CH₃ CN (1 mL each) and eluted three times in 50%CH₃ CN (1 mL each) and washed again twice in 95% CH₃ CN (1 mL each).Almost all of the labeled peptide elutes in the first 50% CH₃ CNelution. This elution is stored at -70° C. and purified by HPLC asneeded for the RIA.

The labeled peptide is purified by reverse phase HPLC on a C8 cartridge(4.6 mm×3 cm, Brownlee). The column is run in a linear gradient from 5%to 45% CH₃ CN in 0.1% TFA in 30 minutes at a flow rate of 0.5 ml/min.Fractions (0.5 mL) are collected and counted. The peak fractioncontaining the labeled peptide is stored at -20° C. and used within 3days in the RIA.

Radioimmunoassay: The buffers used in the RIA are 1) RIA buffer: 0.1MNaPhosphate, pH 7.4 containing 0.1% BSA and 0.1% Triton-X-100. 2) Samplebuffer: 10% Isopropanol in water. 3) Tracer buffer: 0.2M NaPhosphate, pH7.4 containing 0.1% BSA in 0.1% Triton-X-100. The B-amyloid specificantibodies are used at dilutions where approximately 30% of the labeledpeptide is bound in the absence of competing ligand. The dilutions ofthe antibodies are prepared in RIA buffer. The antibodies used in theRIA include three different sera raised to human β-amyloid 1-40synthetic peptide (BA#1, BA#2, and 6514). BA#1 is used at final dilutionof 1/900, BA#2 at 1/1600 and 6514 at 1/2500. The HPLC purified labeledpeptide is diluted in tracer buffer to give between 7000 and 9000 cpm in50 μL. Total displacement is done in the presence of high concentration(2.5 μM) of β-amyloid 1-40. The β-amyloid 1-40 standards are prepared insample buffer. The assay volume is 200 μL. Components are added in thefollowing order:

100 μL Ab in RIA buffer

50 μL Unknown sample or standard or TD in sample buffer

50 μL Labeled peptide (7000-9000 cpm in tracer buffer)

The samples are mixed and incubated overnight at 4° C. To separate thebound counts from the free counts, the assay is terminated withpolyethylene glycol (PEG). To each assay tube, 50 μL of normal rabbitserum is added, followed by 800 μL of PEG (MW6000-8000, 15.8% in RIAbuffer). The samples are incubated for 10 minutes at 4° C. andcentrifuged 3200 rpm, 20 minutes (Sorvall, RT600B). The supernatant isaspirated and the pellets are counted in the gamma counter.

Analysis: Results from antibody binding are interpreted based ondisplacement of the labeled β-amyloid tracer. A positive result is onein which no displacement of tracer is observed, i.e., medium does notcontain secreted β-amyloid indicating the compound tested is effectivein inhibiting β-amyloid production. A negative result is one in whichdisplacement of tracer for antibody binding is seen and equivalent tountreated control cells.

An enzyme linked immunosandwich assay (ELISA) can also be employed toidentify active compounds. Cultured mammalian cells (such as CHO CP-6 orSK-N-MC) producing β-amyloid protein are prepared and treated withcompounds as described for Assay #1 except that radiolabelling of cellprotein is eliminated. Conditioned media from treated cell cultures isharvested and clarified of cellular debris by low-speed centrifugation.The conditioned media is then assayed in a 96 well ELISA formatutilizing -amyloid-specific antibodies. One β-amyloid antibody serves asthe capture reagent for the β-amyloid present in the media samples, thesecond β-amyloid-specific antibody which recognizes a different epitopeon the β-amyloid protein serves as a component of the detector complex.The second β-amyloid antibody is conjugated with biotin which can bedetected by strept-avidin. A third antibody which is coupled tohorseradish peroxidase is used to detect theβ-amyloid:antibody;strept-avidin complex. Addition of o-phenylenediaminesubstrate plus H₂ O₂ and citrate phosphate pH 5 allows for peroxidaseactivity which is quantitated by reading the colorimetric change in themixture at OD^(490nm). Typically, serial three-fold dilutions of eachmedium sample is made in the 96 well plate in addition to a standard,synthetic β-amyloid 1-40 protein. A positive result is one in whichlittle or no reactivity, i.e., adsorbance at OD^(490nm), is obtainedindicated the absence of β-amyloid protein in the medium sample as aresult of inhibition by the compound tested. Partially active inhibitorswould give some but not equivalent adsorbance at OD^(490nm) to a controlmedium sample from untreated cells. Precise quantitation can be achievedby comparing sample values to the standard.

IN VIVO ASSAYS

The activity of the compounds of this invention to prevent or reduce theaccumulation of β-amyloid plaques can be demonstrated in a transgenicmodel of β-amyloid plaque accumulation (e.g., transgenic mouse ortransgenic rat) and in a dog model using dogs with a natural, geneticpredisposition to the formation of β-amyloid plaque. Transgenic micewhich overexpress human β-APP (751) or β-APP (770) in neuronal cells anddisplay histopathology associated with Alzheimer's disease aredescribed, for example, in PCT/US91/04447. In such animal models, thereduction of histopathology and/or symptoms associated with β-amyloidplaque formation such as memory loss, can be used to demonstrate theability of the compounds to treat the therapeutic conditions resultingfrom β-amyloid plaque formation such as Alzheimer's Disease and thememory impairment associated with Down's syndrome.

Since the histopathology in the transgenic mice is more frequent withincreased age of the animal, 2 month old mice would be desirable. The 2month animals would have minimal pathology which would increase withtime in the absence of inhibitory drug. All animals in the experimentwould be from a single pure bred pedigree. One group of mice (n=12)would receive vehicle only; a second group (n=12) would receive a lowdose of drug; a third group (n=12) a moderate dose; and a fourth group(n=12) a high dose. Dosage would be determined from the above assaystaking into account body weight, compound half-life, etc. Ideally, micewould be treated for several months. Delivery of the compound could beby injection, oral route, an implant with timed release, etc., asdictated by the compound profile. Evaluation of treatment would be madeusing immuno-histochemistry to determine the frequency of β-amyloidimmunoreactive deposits in 4 coronal midline sections of brain scored byan investigator blinded from the experimental treatment. Another markerof pathology, Alz50 immunoreactivity, would also be scored for frequencyof occurrence using the same number of brain tissue sections from allmice in the study. A positive result of drug action would be the absenceor reduced frequency of both pathological markers. A physiologicaland/or behavioral correlate unique to the β-amyloid transgenic mice canalso be used to demonstrate drug action.

Some canine races have been reported to have β-amyloid accumulations(Giaccone et al., Neuroscience Letters Vol. 114, pp 178-183 (1990)).Aged non-human primates display β-amyloid pathology, as well as memoryimpairments (Cork et al., American Journal of Pathology, Vol.137, pp1383-1392 (1990)); Podlisny et al., American Journal of Pathology,Vol.138, pp 1423-1425 (1991)). Tests with canines and non-human primateswould most likely follow a somewhat different experimental design withdrug application time being longer.

Any effective amount of a compound of formula 1, or a mixture of morethan one of the compounds of formula 1 may be administered to patient toprevent the abnormal deposition of β-amyloid plaque and to treat adisease or condition associated with the abnormal deposition ofβ-amyloid plaque such as senile dementia of the Alzheimer's type orDown's Syndrome. The specific dosage for preventing the abnormaldeposition of β-amyloid plaque and for treating senile dementia of theAlzheimer's type or Down's Syndrome will depend on factors such as size,type, and age of the patient as well as the severity of the diseasestate or condition, all of which are factors normally familiar to andconsidered by the attending diagnostician treating the patient.Generally, the compounds are administered at a dose of from 0.2 to 20milligrams per kilogram of body weight with a dose of 0.5 to 5 mg/Kgbeing preferred. The compounds can be administered in single or multipleunit dosages containing 25 mg to 250 mg of a compound of formula 1.

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, troches, powders,solutions, suspensions or emulsions. The solid unit dosage forms can bea capsule which can be of the ordinary gelatin type containing, forexample, lubricants and inert filler, such as lactose, sucrose orcornstarch. In another embodiment, the compounds of general formula Ican be tableted with conventional tablet bases such as lactose, sucroseand cornstarch, in combination with binders, such as acacia, cornstarchor gelatin, disintegrating agents such as potato starch or alginic acid,and a lubricant such as stearic acid or magnesium stearate.

For parenteral administration, the compounds may be administered asinjectable dosages of a solution or suspension of the compound in aphysiologically acceptable diluent with a pharmaceutical carrier whichcan be a sterile liquid such as water, alcohols, oils and otheracceptable organic solvents, with or without the addition of asurfactant and other pharmaceutically acceptable adjuvants. Illustrativeof oils which can be employed in these preparations are those ofpetroleum, animal, vegetable, or synthetic origin, for example, peanutoil, soybean oil and mineral oil. In general, water, saline, aqueousdextrose and related sugar solutions, ethanol and glycols such aspropylene glycol or polyethylene glycol, or 2-pyrrolidone are preferredliquid carriers, particularly for injectable solutions.

The compounds can be administered in the form of a depot injection orcerebral implant preparation which may be formulated in such a manner asto permit a sustained release of the active ingredient. The activeingredient can be compressed into pellets or small cylinders andimplanted subcutaneously, intramuscularly, or intracerebrally as depotinjections or implants. Implants may employ inert material such asbiodegradable polymers or synthetic silicones, for example Silastic®, asilicone rubber manufactured by the Dow-Corning Corporation.

The compounds of this invention can also be administered topically. Thiscan be accomplished by simply preparing a solution of the compound to beadministered, preferably using a solvent known to promote transdermalabsorption such as ethanol or dimethyl sulfoxide (DMSO) with or withoutother excipients. Preferably topical administration will be accomplishedusing a patch either of the reservoir and porous membrane type or of asolid matrix variety.

Some suitable transdermal devices are described in U.S. Pat. Nos.3,742,951, 3,797,494, 3,996,934, and 4,031,894. These devices generallycontain a backing member which defines one of its face surfaces, anactive agent permeable adhesive layer defining the other face surfaceand at least one reservoir containing the active agent interposedbetween the face surfaces. Alternatively, the active agent may becontained in a plurality of microcapsules distributed throughout thepermeable adhesive layer. In either case, the active agent is deliveredcontinuously from the reservoir or microcapsules through a membrane intothe active agent permeable adhesive, which is in contact with the skinor mucosa of the recipient. If the active agent is absorbed through theskin, a controlled and predetermined flow of the active agent isadministered to the recipient. In the case of microcapsules, theencapsulating agent may also function as a membrane.

In another device for transdermally administering the compounds inaccordance with the present invention, the pharmaceutically activecompound is contained in a matrix from which it is delivered in thedesired gradual, constant and controlled rate. The matrix is permeableto the release of the compound through diffusion or microporous flow.The release is rate controlling. Such a system, which requires nomembrane is described in U.S. Pat. No. 3,291,636. At least two types ofrelease are possible in these systems. Release by diffusion occurs whenthe matrix is non-porous. The pharmaceutically effective compounddissolves in and diffuses through the matrix itself. Release bymicroporous flow occurs when the pharmaceutically effective compound istransported through a liquid phase in the pores of the matrix.

As is true in many classes of compounds generally suitable for anyparticular pharmacological activity having a therapeutic end-useapplication, certain subgeneric groups and certain specific members ofthe class are preferred because of their overall therapeutic index andtheir biochemical and pharmacological profile. In this instance thepreferred compounds are those wherein P₄ is a bond.

Applicants prefer those compounds wherein X₁ is H or CF₂ C(═O)W. C₁₋₆alkylene is preferably C₁₋₃ alkylene and more preferably methylene orethylene and most preferably branched chain ethylene. Arylalkyl ispreferably benzyl or phenethyl, and aryl is preferably phenyl. The K-P₄-P₃ -P₂ moiety is preferably a protecting group (K) and one amino acid,which is preferably one of the residues of valine, alanine orphenylalanine. R is preferably benzyl, isopropyl or substituted benzylhaving one substituent.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 5                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: unidentified                                                  <220> FEATURE:                                                                <221> NAME/KEY: UNSURE                                                        <222> LOCATION: (1)..(4)                                                       - - <400> SEQUENCE: 1                                                         - - Xaa Xaa Xaa Xaa                                                            1                                                                            - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: unidentified                                                  <220> FEATURE:                                                                <221> NAME/KEY: UNSURE                                                        <222> LOCATION: (1)..(4)                                                       - - <400> SEQUENCE: 2                                                         - - Xaa Xaa Xaa Xaa                                                            1                                                                            - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: unidentified                                                  <220> FEATURE:                                                                <221> NAME/KEY: UNSURE                                                        <222> LOCATION: (1)..(4)                                                       - - <400> SEQUENCE: 3                                                         - - Xaa Xaa Xaa Xaa                                                            1                                                                            - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: unidentified                                                  <220> FEATURE:                                                                <221> NAME/KEY: UNSURE                                                        <222> LOCATION: (1)..(4)                                                       - - <400> SEQUENCE: 4                                                         - - Xaa Xaa Xaa Xaa                                                            1                                                                            - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: unidentified                                                  <220> FEATURE:                                                                <221> NAME/KEY: UNSURE                                                        <222> LOCATION: (1)..(4)                                                       - - <400> SEQUENCE: 5                                                         - - Xaa Xaa Xaa Xaa                                                        __________________________________________________________________________

What is claimed is:
 1. A compound of the formula IB or the hydrate,stereoisomer or pharmaceutically acceptable salt thereof:

    K.sup.a --P.sub.4.sup.a --P.sub.3.sup.a --P.sub.2.sup.a --NH--CH(R.sup.a)--(C(═O)).sub.n --X.sup.a (SEQ ID NO:2)FORMULA IB

wherein X^(a) is CF₂ C(═O)W^(a), wherein W^(a) is arylalkyl, NHCH₂Si(C₁₋₆ alkyl)₂ (Y^(a)),wherein Y^(a) is C₁₋₆ alkyl, C₁₋₆ alkenyl, arylor arylalkyl; n is 1; R^(a) is C₁₋₁₀ alkyl or benzyl; P₂ ^(a) is aresidue of Val, tert-leucine, or Nva; P₃ ^(a) and P₄ ^(a) are eachbonds; and K^(a) is hydrogen, a desamino group, formyl, acetyl,succinyl, benzoyl, t-butyloxycarbonyl, carbobenzyloxy, tosyl, dansyl,isovaleryl, methoxysuccinyl, 1-adamanatanesulphonyl, 1-adamantaneacetyl,2-carboxybenzoyl, phenylacetyl, t-butylacetyl,bis((1-naphthyl)methyl)acetyl, or --A^(a) --R_(z) ^(a) wherein ##STR66##R_(z) ^(a) is an aryl or arylalkyl group in which the aryl groupcontains 6, 10 or 12 carbons suitably substituted by 1 to 3 membersselected independently from the group consisting of fluoro, chloro,bromo, iodo, trifluoromethyl, hydroxy, alkyl containing from 1 to 6carbons, alkoxy containing from 1 to 6 carbons, carboxy,alkylcarbonylamino, wherein the alkyl group contains 1 to 6 carbons,5-tetrazolyl, and acylsulfonamido containing from 1 to 15 carbons,provided that when the acylsulfonamido contains an aryl the aryl may befurther substituted by a member selected from fluoro, chloro, bromo,iodo and nitro; or ##STR67## wherein Z is N or CH, and D^(a) is a groupof the formulae ##STR68## and wherein R'^(a) is hydrogen or a C₁₋₆ alkylgroup provided thatwhen X^(a) is CF₂ C(═O)phenethyl then R^(a) is notbenzyl.
 2. The compound of claim 1 wherein K^(a) is carbobenzyloxy. 3.The compound of claim 1 wherein W^(a) is arylalkyl wherein the alkyl isC₁₋₆ and the aryl is phenyl.
 4. The compound of claim 1 wherein Y^(a) isC₁₋₆ alkyl.
 5. The compound of claim 1 wherein Y^(a) is aryl wherein thearyl is phenyl.
 6. The compound of claim 1 wherein Y^(a) is arylalkylwherein the alkyl is C₁₋₆ and the aryl is phenyl.
 7. The compound ofclaim 1 wherein R^(a) is C₁₋₆ alkyl.
 8. The compound of claim 1 whereinR^(a) is C₁₋₄ alkyl.
 9. The compound of claim 1 wherein W^(a) isarylalkyl, or NHCH₂ Si(C₁₋₆ alkyl)₂ (Y^(a)), wherein aryl is phenyl andthe alkyl is C₁₋₆.
 10. The compound of claim 1 wherein the compound is4-(N-Benzyloxycarbonyl-L-tert-leucyl)amino-2,2-difluoro-3-oxo-5-phenyl-N-trimethylsilymethylpentanamide.
 11. The compound of claim 1 wherein the compound is6-(N-Benzyloxycarbonyl-L-valyl)-amino-4,4-difluoro-1-phenyl-7-methyl-3,5-dioxooctane.12. A pharmaceutical composition comprising an effective amount of acompound of claim 1 and a pharmaceutically acceptable carrier. 13.4-(N-Benzyloxycarbonyl-L-norvalyly)amino-2,2-difluoro-3-oxo-5-phenyl-N-benzylpentanamide. 14.4-(N-Benzyloxycarbonyl-L-tert-leucyl)amino-2,2-difluoro-3-oxo-5-phenyl-N-benzylpentanamide.
 15. A process of making the compounds of formula IB or thehydrate, stereoisomer, isostere or pharmaceutically acceptable saltthereof:

    K.sup.a --P.sub.4.sup.a --P.sub.3.sup.a --P.sub.2.sup.a --NH--CH(R.sup.a)--C(═O)--X.sup.a (SEQ ID NO:5)       FORMULA IB

wherein X^(a) is CF₂ C(═O)W^(a), wherein W^(a) is arylalkyl, NHCH₂Si(C₁₋₆ alkyl)₂ (Y^(a)),wherein Y^(a) is C₁₋₆ alkyl, C₁₋₆ alkenyl, arylor arylalkyl; R^(a) is C₁₋₁₀ alkyl or benzyl; P₂ ^(a) is a residue ofVal, tert-leucine, or Nva; P₃ ^(a) and P₄ ^(a) are each bonds; and K^(a)is hydrogen, a desamino group, formyl, acetyl, succinyl, benzoyl,t-butyloxycarbonyl, carbobenzyloxy, tosyl, dansyl, isovalerylmethoxysuccinyl, 1-adamanatanesulphonyl, 1-adamantaneacetyl,2-carboxybenzoyl, phenylacetyl, t-butylacetyl,bis((1-naphthyl)methyl)acetyl, or --A^(a) --R₂ ^(a) wherein ##STR69##R_(z) ^(a) is an aryl or arylalkyl group in which the aryl groupcontains 6, 10 or 12 carbons suitably substituted by 1 to 3 membersselected independently from the group consisting of fluoro, chloro,bromo, iodo, trifluoromethyl, hydroxy, alkyl containing from 1 to 6carbons, alkoxy containing from 1 to 6 carbons, carboxy,alkylcarbonylamino, wherein the alkyl group contains 1 to 6 carbons,5-tetrazolyl, and acylsulfonamido containing from 1 to 15 carbons,provided that when the acylsulfonamido contains an aryl the aryl may befurther substituted by a member selected from fluoro, chloro, bromo,iodo and nitro; or ##STR70## wherein Z is N or CH, and D^(a) is a groupof the formulae ##STR71## and wherein R'^(a) is hydrogen or a C₁₋₆ alkylgroup; provided thatwhen X^(a) is CF₂ C(═O)phenethyl then R^(a) is notbenzyl; comprising the steps of coupling K^(a) --P₄ ^(a) --P₃ ^(a) --P₂^(a) -- as defined herein to

    H.sub.2 N--CH(R.sup.a)--C(═O)X.sup.a

as defined herein, and optionally obtaining the pharmaceuticallyacceptable salt thereof, or alternativelycoupling K^(a) --P₄ ^(a) --P₃^(a) --P₂ ^(a) -- as defined herein to

    H.sub.2 N--CH(R.sup.a)--CH(OH)X.sup.a

as defined herein, and oxidizing the so-produced compound to obtain

    K.sup.a --P.sub.4.sup.a --P.sub.3.sup.a --P.sub.2.sup.a --NH--CH(R.sup.a)--C(═O)--X.sup.a (SEQ ID NO:5)       FORMULA IB

optionally obtaining the pharmaceutically acceptable salt thereof.
 16. Amethod for treating a patient for senile demnentia of the Alzheimer'stype by administering to a patient in need thereof a therapeuticallyeffective amount of a compound of formula IB or the hydrate,stereoisomer, isostere or pharmaceutically acceptable salt thereof:

    K.sup.a --P.sub.4.sup.a --P.sub.3.sup.a --P.sub.2.sup.a --NH--CH(R.sup.a)--C(═O)--X.sup.a (SEQ ID NO:5)       FORMULA IB

whereinX^(a) is CF₂ C(═O)W^(a), wherein W^(a) is arylalkyl, NHCH₂Si(C₁₋₆ alkyl)₂ (Y^(a)),wherein Y^(a) is C₁₋₆ alkyl, C₁₋₆ alkenyl, arylor arylalkyl; R^(a) is C₁₋₁₀ alkyl or benzyl; P₂ ^(a) is a residue ofVal, tert-leucine, or Nva; P₃ ^(a) and P₄ ^(a) are each bonds; and K^(a)is hydrogen, a desamino group, formyl, acetyl, succinyl, benzoyl,t-butyloxycarbonyl, carbobenzyloxy, tosyl, dansyl, isovaleryl,methoxysuccinyl, 1-adamanatanesulphonyl, 1-adamantaneacetyl,2-carboxybenzoyl, phenylacetyl, t-butylacetyl,bis((1-naphthyl)methyl)acetyl, or --A^(a) --R_(z) ^(a) wherein ##STR72##R_(z) ^(a) is an aryl or arylalkyl group in which the aryl groupcontains 6, 10 or 12 carbons suitably substituted by 1 to 3 membersselected independently from the group consisting of fluoro, chloro,bromo, iodo, trifluoromethyl, hydroxy, alkyl containing from 1 to 6carbons, alkoxy containing from 1 to 6 carbons, carboxy,alkylcarbonylamino, wherein the alkyl group contains 1 to 6 carbons,5-tetrazolyl, and acylsulfonamido containing from 1 to 15 carbons,provided that when the acylsulfonamido contains an aryl the aryl may befurther substituted by a member selected from fluoro, chloro, bromo,iodo and nitro; or ##STR73## wherein Z is N or CH, and D^(a) is a groupof the formulae ##STR74## and wherein R'^(a) is hydrogen or a C₁₋₆ alkylgroup; provided that when X^(a) is CF₂ C(═O)phenethyl then R^(a) is notbenzyl.